Therapeutic and diagnostic methods and compositions based on jagged/notch proteins and nucleic acids

ABSTRACT

This invention relates to therapeutic and diagnostic methods and compositions based on Jagged/Notch proteins and nucleic acids, and on their role in the signaling pathway relating to endothelial cell migration and/or differentiation. In addition, this invention provides a substantially purified Jagged protein, as well as a substantially purified nucleic acid or segment thereof encoding Jagged protein, or a functionally equivalent derivative, or allelic or species variant thereof. Further, this invention provides a substantially purified soluble Jagged protein and a substantially purified nucleic acid encoding same as well as a recombinant cell comprising a nucleic acid encoding a soluble Jagged protein. Soluble Jagged provides further therapeutic and diagnostic methods relating to diseases, disorders, and conditions involving Jagged/Notch signaling including, inter alia, angiogenesis, differentiation, and control of gene expression.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. applicationSer. No. 09/199,865, now U.S. Pat. No. 6,433,138 filed on Nov. 25, 1998,which is a continuation of PCT Application No. US/PCT97/09407, filed onMay 30, 1997, all of which are entitled to priority under 35 U.S.C.§119(e), to U.S. Provisional Application No. 60/018,841, filed on May31, 1996, and all of which are hereby incorporated herein by referencein their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was supported in part by funds from the U.S. Government(National Institutes of Health Grant Nos. AG07450-12, HL32348-18,HL54710-04, and HL35627-16) and the U.S. Government may therefore havecertain rights in the invention.

BACKGROUND OF THE INVENTION

The present invention relates to therapeutic and diagnostic methods andcompositions based on Jagged/Notch proteins and nucleic acids, and onthe role of their signaling pathway in endothelial cell migration,angiogenesis, and/or differentiation.

The functional integrity of the human vascular system is maintained bythe endothelial cell which monitors the non-thrombogenic interfacebetween blood and tissue in vivo. Thus, factors that influence humanendothelial cell function may contribute significantly to the regulationand maintenance of homeostasis (see Maciag, 1984, In: Progress inHemostasis and Thrombosis, pp. 167-182, Spaet, ed., A. R. Liss, NewYork; Folkman and Klagsbum, 1987, Science 235:442-447; Burgess andMaciag, 1989, Annu. Rev. Biochem. 58:575-606). Likewise, events thatperturb this complex equilibrium are relevant to the pathophysiology ofhuman disease states in which cellular components of the vascular treeare active participants including, e.g., atherogenesis, coronaryinsufficiency, hypertension, rheumatoid arthritis, solid tumor growthand metastasis, and wound repair.

Since the endothelium is present in all organs and tissues, endothelialcell function is also fundamental to the physiology and integration ofthese multicellular systems. This includes the ability to monitor andinterface with repair systems that employ the tightly regulatedinflammatory, angiogenic and neurotropic responses. Indeed, biochemicalsignals that are responsible for the modification of these responseshave been well characterized as polypeptide growth factors andcytokines; however, their mechanisms of operation have, prior to thepresent invention, been poorly understood, impeding their acceptance asvaluable tools in clinical management.

A major accomplishment of modern biology has been the recognition thatstructural elements responsible for physiologic functions are conservedthroughout the animal kingdom. Genetic analysis of yeast, C. elegans,Xenopus, Zebra fish, and Drosophila, among others, has provided newinsight into the regulation of the cell cycle, organelle biosynthesisand trafficking, cell fate and lineage decisions during development, aswell as providing the fundamental principles fortranscriptional/translational/post-translational regulation. Indeed, theconservation of structure-function principles exhibited by such systemshas generated new insight into these and other regulatory systemsutilized by mammalian cells. Moreover, a resolution of the geneticstructure of the mammalian homologs for such genes in non-mammalianspecies has often led to a discernment of their function in mammals,even though the delineation of the function of a particular homologousmammalian gene or gene fragment may well be serendipitous. In manycases, it is the result produced by expression and differential cDNAcloning strategies that manifest mammalian DNA sequences with homologyto genes previously identified in more primitive species.

During the past decade, differential cDNA cloning methods, includinge.g., conventional subtractive hybridization (Hla and Maciag, 1990,Biochem. Biophys. Res. Commun. 167:637-643), differential polymerasechain reaction (PCR)-oriented hybridization (Hla and Maciag, 1990, J.Biol. Chem. 265:9308-9313), and more recently, a modification of thedifferential display (Zimrin et al., 1995, Biochem. Biophys. Res.Commun. 213:630-638) were used to identify genes induced during theprocess of human umbilical vein endothelial cell (HUVEC) differentiationin vitro. Very early studies disclosed that HUVEC populations are ableto generate capillary-like, lumen-containing structures when introducedinto a growth-limited environment in vitro (Maciag et al., 1982, J. CellBiol. 94:511-520). These studies permitted the identification andcharacterization of protein components of the extracellular matrix asinducers of this differentiation process, while at the same timedefining the capillary-like structures as non-terminally differentiated(Maciag, 1984, In: Progress in Hemostasis and Thrombosis, pp. 167-182,Spaet, ed., A. R. Liss, New York). Additional experiments haveelucidated the importance of polypeptide cytokines, such as IL-1 (Maieret al., 1990, J. Biol. Chem. 265:10805-10808) and IFNγ (Friesel et al.,1987, J. Cell Biol. 104:689-696), as inducers of HUVEC differentiationin vitro, and ultimately lead to an understanding that the precursorform of IL-1α was responsible for the induction of HUVEC senescence invitro (Maciag et al., 1981, J. Cell Biol. 91:420-426; Maier et al.,1990, Science 249:1570-1574)—the only truly terminal HUVEC phenotypeidentified to date as summarized in FIG. 1.

Recent research has employed differential cDNA cloning methods, whichpermits the identification of new and very interesting genes. However,until very recently, establishing their identity did not provide insightinto the mechanism of HUVEC differentiation. Current research hasfocused upon the fibroblast growth factor (FGF) and interleukin (IL)-1gene families as regulators of the angiogenesis process, both in vitroand in vivo (Friesel et al., 1995, FASEB J. 9:919-925; Zimrin et al.,1996, J. Clin. Invest. 97:1359). The human umbilical vein endothelialcell (HUVEC) has proven to be an effective model for studying the signalpathways utilized by FGF-1 to initiate HUVEC migration and growth, therole of IL-1α as an intracellular inhibitor of FGF-1 function andmodifier of HUVEC senescence, and the interplay between the FGF and theIL-1 gene families as key effectors of HUVEC differentiation in vitro.Such insight has enabled the present inventors to use modem molecularmethods to identify a key regulatory ligand-receptor signaling system,which is able to both induce capillary endothelial cell migration andrepress large vessel endothelial cell migration.

The Jagged/Serrate/Delta-Notch/Lin/Glp signaling system, originallydescribed during the development of C. elegans and Drosophila as anessential system instrumental in cell fate decisions, has been found tobe highly conserved in mammalian cells (Nye and Kopan, 1995, Curr. Biol.5:966-969). Notch proteins comprise a family of closely-relatedtransmembrane receptors initially identified in embryologic studies inDrosophila (Fortini and Artavanis-Tsakonas, 1993, Cell 75:1245-1247).The genes encoding the Notch receptor show a high degree of structuralconservation, and contain multiple EGF repeats in their extracellulardomains (Coffman et al., 1990, Science 249:1438-1441; Ellisen et al.,1991, Cell 66:649-661; Weinmaster et al., 1991, Development 113:199-205;Weinmaster et al., 1992, Development 116:931-941; Franco del Amo et al.,1992, Development 115:737-744; Reaume et al., 1992, Dev. Biol.154:377-387; Lardelli and Lendahi, 1993, Mech. Dev. 46:123-136; Bierkampand Campos-Ortega, 1993, Mech. Dev. 43:87-100; Lardelli et al., 1994,Exp. Cell Res. 204:364-372). In addition to the thirty-six EGF repeatswithin the extracellular domain of Notch 1, there is a cys-rich domaincomposed of three Notch Lin Glp (NLG) repeats, which is important forligand function, followed by a cys-poor region between the transmembraneand NLG domain.

The intracellular domain of Notch 1 contains six ankyrin/Cdc 10 repeatspositioned between two nuclear localization sequences (NLS)(Artavanis-Tsakonas et al., 1995, Science 268:225-232). This motif isfound in many functionally diverse proteins (see, e.g., Bork, 1993,Proteins 17:363-374), including members of the Rel/NF-κB family (Blanket al., 1992, TIBS 17:135-140), and is thought to be responsible forprotein-protein interactions. Notch has been shown to interact with anovel ubiquitously distributed cytoplasmic protein deltex through itsankyrin repeats, a domain shown by deletion analysis to be necessary foractivity (Matsuno et al., 1995, Development 121:2633-2644).

Carboxy terminal to this region is a polyglutamine-rich domain (OPA) anda pro-glu-ser-thr (PEST) domain (SEQ ID NO:33) which may be involved insignaling protein degradation. There are numerous Notch homologs,including three Notch genes. (The corresponding structures for Lin-12and Glp-1 are shown in FIG. 4.)

Several Notch ligands have been identified in vertebrates, includingDelta, Serrate and Jagged. The Notch ligands are also transmembraneproteins, having highly conserved structures. These ligands are known tosignal cell fate and pattern formation decisions through the binding tothe Lin-12/Notch family of transmembrane receptors (Muskavitch andHoffmann, 1990, Curr. Top. Dev. Biol. 24:289-328; Artavanis-Tsakonas andSimpson, 1991, Trends Genet. 7:403-408; Greenwald and Rubin, 1992, Cell68:271-281; Gurdon, 1992, Cell 68:185-199; Fortini andArtavanis-Tsakonas, 1993, Cell 75:1245-1247; and Weintraub, 1993, Cell75:1241-1244). A related protein, the Suppressor of hairless (Su(H)),when co-expressed with Notch in Drosophila cells, is sequestered in thecytosol, but is translocated to the nucleus when Notch binds to itsligand Delta (Fortini and Artavanis-Tsakonas, 1993, Cell 75:1245 -1247).Studies with constitutively activated Notch proteins missing theirextracellular domains have shown that activated Notch suppressesneurogenic and mesodermal differentiation (Coffinan et al., 1993, Cell73:659-671; Nye et al., 1994, Development 120:2421-2430).

The Notch signaling pathway (FIG. 3), which is apparently activated byJagged in the endothelial cell, involves cleavage of the intracellulardomain by a protease, followed by nuclear trafficking of the Notchfragment and the interaction of this fragment with the KBF₂/RBP-J_(k)transcription factor (Jarriault et al., 1995, Nature 377:355-358; Kopanet al., 1996, Proc. Natl. Acad. Sci. USA 93:1683-1688), a homolog of theDrosophila Suppressor of hairless gene (Schweisguth et al., 1992, Cell69:1199-1212), a basic helix-loop-helix transcription factor involved inNotch signaling in insects (Jennings et al., 1994, Development120:3537-3548) and in the mouse (Sasai et al., 1992, Genes Dev.6:2620-2634). This effector is able to repress the transcriptionalactivity of other genes encoding transcription factors responsible forentry into the terminal differentiation program (Nye et al., 1994; Kopanet al., 1994, J. Cell. Physiol. 125:1-9).

The Jagged gene encodes a transmembrane protein which is directed to thecell surface by the presence of a signal peptide sequence (Lindsell etal., 1995, Cell 80:909-917). While the intracellular domain contains asequence with no known homology to intracellular regions of othertransmembrane structures, the extracellular region of the ligandcontains a cys-rich region, 16 epidermal growth factor (EGF) repeats,and a DSL (Delta Serrate Lag) domain. As shown in FIG. 2, the DSL domainas well as the EGF repeats, are found in other genes including theDrosophila ligands, Serrate (Baker et al., 1990, Science 250:1370-1377;Thomas et al., 1991, Development 111:749-761) and Delta (Kopczynski etal., 1988, Genes Dev. 2:1723 -1735), and C. elegans genes Apx-1(Henderson et al., 1994, Development 120:2913-2924; Mello et al., 1994,Cell 77:95-106) and Lag-2 (Tax et al., 1994, Nature 368:150-154).

Nevertheless, until the discovery of the presently disclosed invention,human Jagged remained undefined and the function and relationship, ifany, of the human ligand to Notch remained unknown in the art. However,there was a recognized need in the art for a complete understanding ofthe protein's role in the regulation of cell differentiation andregulation. The present invention provides this understanding and inaddition, provides compositions and methods useful for treatment ofJagged-related diseases in mammals.

BRIEF SUMMARY OF THE INVENTION

The invention includes an isolated nucleic acid encoding a solubleJagged protein. The invention also includes a vector and a recombinantcell comprising the isolated nucleic acid. Further, the inventionincludes an isolated polypeptide encoded by isolated nucleic acid.

In one aspect, the nucleic acid comprises a portion of sequence of SEQID NO:2, where the portion comprises the soluble Jagged.

The invention also includes an isolated nucleic acid having at least 30%identity with from about nucleotide number I to about nucleotide 3201 ofSEQ ID NO:2.

The invention includes an isolated nucleic acid encoding a solubleJagged protein, the nucleic acid having at least about 20% identity withSEQ ID NO:17. In one aspect, the nucleic acid has the sequence of SEQ IDNO:17.

The invention includes an isolated nucleic acid encoding a solubleJagged protein, the soluble Jagged protein having at least about 40%identity with SEQ ID NO:18. The invention further includes a vector anda recombinant cell comprising this isolated nucleic acid. The inventionalso includes an isolated polypeptide encoded by the nucleic acid.

In one aspect, the nucleic acid encoding a soluble Jagged protein hasthe sequence of SEQ ID NO:18.

The invention includes an isolated nucleic acid encoding a solubleJagged protein, where the nucleic acid further comprises a nucleic acidencoding a tag polypeptide covalently linked thereto.

In one aspect, the tag polypeptide is selected from the group consistingof a myc tag polypeptide, a myc-pyruvate kinase tag polypeptide, aglutathione-S-transferase tag polypeptide, a maltose binding tagpolypeptide, green fluorescence protein tag polypeptide, an alkalinephosphatase tag polypeptide, a His6 tag polypeptide, an influenza virushemagglutinin tag polypeptide, and a maltose binding protein tagpolypeptide.

In another aspect, the tag polypeptide is a myc tag polypeptide.

The invention includes an isolated nucleic acid encoding a solubleJagged protein, where the nucleic acid further comprises apromoter/regulatory sequence operably linked thereto.

The invention includes an isolated soluble Jagged polypeptide. In oneaspect, the isolated polypeptide shares at least about 20% identity witha polypeptide having the amino acid sequence of SEQ ID NO:18. In afurther aspect, the polypeptide is SEQ ID NO:18.

The invention includes an isolated polypeptide encoded by an isolatednucleic acid encoding a soluble Jagged, where the polypeptide has atleast about 20% identity with from about amino acid residue 1 to aboutamino acid residue 1067 of the sequence of SEQ ID NO:1.

In one aspect, the polypeptide further comprises a tag polypeptide. In afurther aspect, the tag polypeptide is selected from the groupconsisting of a myc tag polypeptide, a myc-pyruvate kinase tagpolypeptide, a glutathione-S-transferase tag polypeptide, a maltosebinding tag polypeptide, green fluorescence protein tag polypeptide, analkaline phosphatase tag polypeptide, a His6 tag polypeptide, aninfluenza virus hemagglutinin tag polypeptide, and a maltose bindingprotein tag polypeptide. In yet a further aspect, the tag epitope is amyc tag epitope.

The invention includes a recombinant cell comprising an isolatedpolypeptide encoded by an isolated nucleic acid encoding a solubleJagged protein.

The invention includes a composition comprising an isolated solubleJagged polypeptide in a pharmaceutically acceptable carrier.

The invention also includes a composition comprising a nucleic acidencoding a soluble Jagged protein in a pharmaceutically acceptablecarrier.

The invention includes a pharmaceutical composition comprising atherapeutically effective amount of an isolated nucleic acid encoding asoluble Jagged polypeptide, or a functionally equivalent derivative, oran allelic or species variant thereof, in a pharmaceutically acceptablecarrier.

The invention further includes a pharmaceutical composition comprising atherapeutically effective amount of an isolated soluble Jaggedpolypeptide, or a functionally equivalent derivative, or an allelic orspecies variant thereof, in a pharmaceutically acceptable carrier.

The invention includes a pharmaceutical composition comprising arecombinant cell comprising an isolated nucleic acid encoding a solubleJagged polypeptide in a pharmaceutically acceptable carrier.

The invention also includes a pharmaceutical composition comprising arecombinant cell comprising an isolated soluble Jagged polypeptide.

The invention includes a method of affecting angiogenesis in a systemcapable of angiogenesis. The method comprises contacting a cell with anangiogenic effective amount of an isolated soluble Jagged polypeptide,thereby affecting angiogenesis in a system capable of angiogenesis.

The invention includes a method of affecting angiogenesis in a mammal.The method comprises administering to a mammal an angiogenic effectiveamount of an isolated soluble Jagged polypeptide, thereby affectingangiogenesis in a mammal. In one aspect, the isolated soluble Jaggedpolypeptide is administered by administering to the mammal at least onemolecule selected from the group consisting of an isolated solubleJagged polypeptide, an isolated nucleic acid encoding a soluble Jaggedpolypeptide, and a recombinant cell comprising an isolated nucleic acidencoding a soluble Jagged polypeptide.

The invention also includes a method of affecting differentiation of acell. The method comprises contacting a cell with a differentiationeffective amount of an isolated soluble Jagged polypeptide, therebyaffecting differentiation of said cell. In one aspect, the cell isselected from the group consisting of a mesodermal-derived cell, anendodermal-derived cell, an ectodermal-derived cell, and aneurodermal-derived cell.

The invention includes a method of identifying a compound capable ofaffecting differentiation of a cell. The method comprises contacting arecombinant cell comprising an isolated nucleic acid encoding a solubleJagged protein expressed therefrom with a test compound and comparingthe growth characteristics of the cell contacted with the compound withthe growth characteristics of an otherwise identical cell not contactedwith the compound, wherein a difference in the growth characteristics ofthe cell contacted with the compound compared with the growthcharacteristics of the otherwise identical cell not contacted with thecompound is an indication that the compound is capable of affectingdifferentiation of the cell.

The invention includes a method of identifying a compound capable ofaffecting the binding of Jagged ligand to a Notch receptor. The methodcomprises contacting a recombinant cell comprising a nucleic acidencoding a soluble Jagged protein with a test compound and comparing thegrowth characteristics of the cell contacted with the compound with thegrowth characteristics of an otherwise identical cell not contacted withthe compound, wherein a difference in the growth characteristics of thecell contacted with the compound compared with the growthcharacteristics of the otherwise identical cell not contacted with thecompound is an indication that the compound is capable of affecting thebinding of Jagged ligand to a Notch receptor.

The invention includes a method of identifying a compound capable ofaffecting angiogenesis. The method comprises contacting a recombinantcell comprising a nucleic acid encoding a soluble Jagged proteinexpressed therefrom with a test compound and comparing the growthcharacteristics of the cell contacted with the compound with the growthcharacteristics of an otherwise identical cell not contacted with thecompound, wherein a difference in the growth characteristics of the cellcontacted with the compound compared with the growth characteristics ofthe otherwise identical cell not contacted with the compound is anindication that the compound is capable of affecting angiogenesis.

The invention further includes a method of inhibiting expression of typeI collagen in a cell. The method comprises administering an expressioninhibiting amount of soluble Jagged to a cell, thereby inhibitingexpression of type I collagen.

In one aspect, the soluble Jagged is administered as a substanceselected from the group consisting of an isolated nucleic acid encodingsoluble Jagged, a vector expressing soluble Jagged, and an isolatedsoluble Jagged polypeptide.

The invention includes a kit for affecting angiogenesis in a mammal. Thekit comprises an angiogenic effective amount of an isolated solubleJagged polypeptide, an applicator, and an instructional material for theuse of the kit.

The invention includes a kit for affecting differentiation of a cell.The kit comprises a differentiation effective amount of an isolatedsoluble Jagged polypeptide, an applicator, and an instructional materialfor the use of the kit.

The invention includes a kit for inhibiting expression of type Icollagen in a cell. The kit comprises an expression inhibiting amount ofsoluble Jagged, an applicator, and an instructional material for the useof the kit.

Additional objects, advantages and novel features of the invention willbe set forth in part in the description which follows, and in part willbecome apparent to those skilled in the art on examination of thefollowing, or may be learned by practice of the invention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a diagram illustrating the phenotypic alterations of HUVEC bycytokines. Early studies demonstrated that HUVEC populations are able togenerate capillary-like, lumen-containing structures when introducedinto a growth-limited environment in vitro. However, exposure of anHUVEC population to polypeptide cytokines, such as IL-1 and IFNγ, asinducers of HUVEC differentiation in vitro, led to an understanding thatthe precursor form of IL-1α was responsible for the induction of HUVECsenescence in vitro, the only truly terminal HUVEC phenotype identifiedto date. (PD=population doubling).

FIG. 2 is a diagram illustrating the domain structure of the Notchligand family. (Numbers refer to the number of EGF repeats in theextracellular domain.) As indicated in this chart, although theintracellular domain of the Jagged gene contains a sequence with noknown homology to intracellular regions of other transmembranestructures, the extracellular region of the gene contains a cys-richregion, 16 epidermal growth factor (EGF) repeats, and aDelta-Serrate-Lag (DSL) domain, typical of comparable regions found inother genes including the Drosophila ligands, Serrate and Delta, and theC. elegans genes, Apx-1 and Lag-2.

FIG. 3 is a diagram illustrating the Notch signaling pathway. Thecomponents of the Notch signaling pathway are illustrated, using themyoblast as an example. The Notch signaling pathway, when activated byJagged in the endothelial cell, involves cleavage of the intracellulardomain by a protease, nuclear trafficking of the Notch fragment and theinteraction of this fragment with the KBF₂/RBP-Jk transcription factor,a homolog of the Drosophila Suppressor of Hairless (Su(H)) gene, whichis a basic helix-loop-helix transcription factor involved in Notchsignaling.

FIG. 4 is a diagram illustrating the domain structure of the Notchreceptor family. (Numbers refer to the number of EGF repeats in theextracellular domain.) As indicated in this chart, in addition to the 36EGF repeats within the extracellular domain of Notch 1, there is acys-rich domain composed of three Notch-Lin-Glp (NLG) repeats, followedby a cys-poor region between the transmembrane and NLG domain. Theintracellular domain of Notch 1 contains six ankyrin/Cdc10 repeatspositioned between two nuclear localization sequences (NLS). In thecarboxy-terminal direction from this region is a polyglutamine-richdomain (OPA) and a pro-glu-ser-thr (PEST) domain (SEQ ID NO:33):Comparable structures are shown for Lin-12 and Glp-1.

FIG. 5 is an image of a gel depicting the RT-PCR analysis ofsteady-state levels of Jagged, Notch I and Notch 2 transcripts in HUVEC.Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a positivecontrol.

FIG. 6 is a graph depicting the effect of the Jagged antisenseoligonucleotide (JAS) (5′-TGGGGACCGCATCGCTGC-3′ [SEQ ID NO:29]) on BMECsprout formation, as compared with the effect on three controloligomers, a Jagged sense oligonucleotide (JS) (5′-GCAGCGATGCGGTCCCCA-3′[SEQ ID NO:30]), a 3′ antisense Jagged oligomer (3′ AS)(5′-GAATCAAGGCTCCCCTAG-3′ [SEQ ID NO:31]), and a mutated 5′ antisenseJagged (MUT5′ AS) oligomer (5′-TGCGGTCCCCAACGGTGG-3′ [SEQ ID NO:32]).

FIG. 7A is a graph depicting the effect of the antisense Jaggedoligonucleotide on bovine microvascular endothelial cells (BMEC).

FIG. 7B is a graph depicting the effect of the antisense Jaggedoligonucleotide on bovine aorta endothelial cells (BAEC).

FIG. 8A is a diagram depicting the amino acid sequence of human Jagged(GenBank Accession No. U77720 [SEQ ID NO:1]). The amino acid sequence,which is depicted using the standard one-letter amino acid residue code,is provided. The amino acid sequence comprises various domainsincluding, but not limited to, a signal peptide (from about amino acidresidue 1 to about amino acid residue 21); a DSL domain (from aboutamino acid residue 185 to about amino acid residue 229); EGF repeats(from about amino acid residue 234 to about amino acid residue 862); acysteine-rich region (from about amino acid residue 863 to about aminoacid residue 1002); a transmembrane domain (from about amino acidresidue 1068 to about amino acid residue 1093); and a cytoplasmic region(from about amino acid residue 1094 to about amino acid residue 1218).

FIGS. 8B-C is a diagram depicting the nucleic acid sequence of humanJagged (GenBank Acc. No. U77720 [SEQ ID NO:2]). Nucleotides designatedby “Y” indicates C or T at that position, and nucleotides designated by“R” indicates G or A.

FIG. 9 is an image depicting an immunoblot analysis of murine pro-α-1(I)collagen expression in insert-less vector and soluble Jagged-1 NIH 3T3cell transfectants. Cell lysates were prepared from pMexNeo insert-lessvector control NIH 3T3 cell transfectants (lane 1), and soluble Jagged-1NIH 3T3 transfectant clones 38-1 (lane 2) and 38-4 (lane 3). Theproteins were transferred to Hybond C membranes and the blots wereimmunostained using SP1.D8 monoclonal antibody specific for thepro-α-1(I) collagen amino-terminal extension peptide as describedelsewhere herein.

FIG. 10A is an image depicting the growth of control emptyvector-transfected NIH 3T3 cells on plastic. Empty vector-transfectedcontrol NIH 3T3 cells were plated at 2×10⁴ cells per cm² on cell cultureplastic. Two days after plating, the empty vector-transfected NIH 3T3cells on plastic did not form multicellular chords. (Phase contrast at amagnification of 100×).

FIG. 10B is an image depicting formation of multicellular chords ofsoluble Jagged-1 transfected NIH 3T3 cells on plastic. Soluble Jagged-1transfected NIH 3T3 cells were plated at 2×10⁴ cells per cm² on cellculture plastic. Two days after plating, the soluble Jagged-1transfectants formed multicellular chords on plastic. (Phase contrast ata magnification of 100×).

FIG. 10C is an image depicting growth of control emptyvector-transfected NIH 3T3 cells on collagen. Empty vector-transfectedcontrol NIH 3T3 cells were plated at 2×10⁴ cells per cm² on collagen.Two days after plating, the empty vector-transfected control NIH 3T3cells did not form multicellular chords on collagen. (Phase contrast ata magnification of 100×).

FIG. 10D is an image depicting formation of chords by soluble Jagged-1transfected NIH 3T3 cells grown on collagen. Soluble Jagged-1transfected NIH 3T3 cells were plated at 2×10⁴ cells per cm² oncollagen. Two days after plating, the soluble Jagged-1 transfectantsformed multicellular chords on both plastic (FIG. 10B, supra) and oncollagen. (Phase contrast at a magnification of 100×).

FIG. 11 is a graph depicting the growth kinetics of soluble Jagged-1 andcontrol insert-less vector NIH 3T3 cell transfectants. The cells wereplated at a seed density of 1×10⁴ cells per cm² and the cell numberswere assessed daily in quadruplicate via hemocytometer count. Bothinsert-less vector and soluble Jagged-1 cell populations reachedconfluence at approximately 4 days after plating. The data disclosed arethe mean±standard error of the mean.

FIG. 12A is an image depicting the angiogenesis present in tissues ofnude mice injected with soluble Jagged-1 transfected NIH 3T3 cells. Theimage depicts soluble Jagged-1 tissue mass formation in nude mice. Theimage depicts a deep dermal view of a soluble Jagged-1 NIH 3T3 celltissue mass 10 weeks after intradermal injection of the celltransfectants into the flank of a nude mouse. The data discloseddemonstrate prominent angiogenesis and arborizing microvessels over thedeep surface.

FIG. 12B is an image depicting the angiogenesis present in tissues ofnude mice injected with soluble Jagged-1 transfected NIH 3T3 cells. Theimage depicts hematoxylin and eosin staining of a paraffin section ofthe soluble Jagged-1 tissue mass depicted in FIG. 12A. The image depictsprominent surface blood-filled capillaries, penetrating vessels, andintra-tumor blood islands. Magnification is 100×.

FIG. 12C is an image depicting the immunohistochemical analysis oftissues of nude mice injected with soluble Jagged-1 transfected NIH 3T3cells using anti-CD31 (PECAM) antibody. The image depicts a lowmagnification (100×) view of a frozen section of the tissue massdepicted in FIG. 12A demonstrating the immunohistochemical localizationof CD31 (PECAM). The image depicts two cross sections of a microvesselalong with a high density of CD31 positivity.

FIG. 12D is an image depicting the immunohistochemical analysis oftissues of nude mice injected with soluble Jagged-1 transfected NIH 3T3cells using anti-CD31 (PECAM) antibody. The image depicts a highermagnification (500×) of the view of a frozen section of the tissue masswhich is depicted at a magnification of 100× in FIG. 12C. The datadisclosed herein demonstrate that immunohistochemical localization ofCD31 is comprised of groups of single cells or angulated collection ofCD31-positive cells.

FIG. 13A is an image depicting the amino acid sequence of soluble-Jagged(SEQ ID NO:18).

FIGS. 13B-C is an image depicting the nucleic acid sequence ofsoluble-Jagged (SEQ ID NO:17). Nucleotides designated by “Y” indicates Cor T at that position, and nucleotides designated by “R” indicates G orA.

DETAILED DESCRIPTION OF THE INVENTION

As disclosed in the present invention, the human Jagged gene (andsoluble forms thereof) has now been cloned, isolated and defined, andthe Jagged-Notch role in endothelial cell differentiation and/ormigration has been elucidated. In addition, it is presently disclosedthat the novel signaling pathway produces disparate effects on themigration of large and small vessel endothelial cells, providing whatappears to be the first demonstration of a signaling difference betweenlarge and small vessel endothelial cells both in degree and direction.This highlights the potential function of a previously unknownligand-receptor signaling pathway in the endothelial cell which ismodulated during the migratory phase of angiogenesis. Moreover, thepresent invention provides an explanation of the previously unresolvedphenomenon in which endothelial cells have been shown to reproduciblydifferentiate into a non-terminal and completely reversible tubular-likecell phenotype in vitro (Maciag et al., 1982, J. Cell Biol. 94:511-520).Thus, the present invention significantly advances the art, providingnot only methods of regulating cell differentiation and angiogenesis,but also teaching a method for preventing the undesirable migration ofspecific cell types into large blood vessels following angioplasticsurgery to control restenosis.

Definitions

As used herein, each of the following terms has following meaning.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, the term “adjacent” is used to refer to nucleotidesequences which are directly attached to one another, having nointervening nucleotides. By way of example, the pentanucleotide5′-AAAAA-3′ is adjacent the trinucleotide 5′-TTT-3′ when the two areconnected thus: 5′-AAAAATTT-3′ or 5′-TTTAAAAA-3′, but not when the twoare connected thus: 5′-AAAAACTTT-3′.

As used herein, amino acids are represented by the full name thereof, bythe three letter code corresponding thereto, or by the one-letter codecorresponding thereto, as indicated in the following table:

Full Name Three-Letter Code One-Letter Code Aspartic Acid Asp D GlutamicAcid Glu E Lysine Lys K Arginine Arg R Histidine His H Tyrosine Tyr YCysteine Cys C Asparagine Asn N Glutamine Gln Q Serine Ser S ThreonineThr T Glycine Gly G Alanine Ala A Valine Val V Leucine Leu L IsoleucineIle I Methionine Met M Proline Pro P Phenylalanine Phe F Tryptophan TrpW

By the term “angiogenic effective amount,” as the term is used herein,is meant an amount of soluble Jagged, or a mutant, derivative, variant,or fragment thereof, which when administered to a cell, tissue, ororganism, induces a detectable increase in the level of angiogenesis inthe cell, tissue, or organism, compared with the level of angiogenesisprior to or on the absence of the administration of the soluble Jagged.

“Angiogenesis,” as used herein, means the formation of new blood vesselsand encompasses the development of angiogenic tissue and/or altered cellor tissue morphology typical of angiogenic tissue development. Oneskilled in the art would appreciate, based upon the disclosure providedherein, that the level of angiogenesis can be assessed using, forexample but not limited to, a CAM assay, a nude mouse in vivo assay, anendothelial cell migration assay to assess sprout formation, thedevelopment of chord-like structures, and the like.

By the term “angiogenesis effective amount,” as used herein, is meant anamount of soluble Jagged that mediates a detectable increase or decreasein the level of angiogenesis in a cell, tissue, or organism. One skilledin the art would appreciate, based upon the disclosure provided herein,that such amount depends on the nature of the cell, tissue or organismto which the soluble Jagged is administered. The skilled artisan wouldfurther appreciate, based upon the disclosure provided herein, thatthere are a number of assays, several of which are disclosed elsewhereherein, useful for assessing the level of angiogenesis in a cell, atissue, and/or an organism, and such assays, as well as those developedin the future, are contemplated in the present invention.

By the term “applicator” as the term is used herein, is meant any deviceincluding, but not limited to, a hypodermic syringe, a pipette, and thelike, for administering the soluble Jagged nucleic acid, protein, and/orcomposition of the invention to a mammal.

“Antisense nucleic acid sequence,” “antisense sequence,” “antisense DNAmolecule” or “antisense gene” refer to pseudogenes which are constructedby reversing the orientation of the gene with regard to its promoter, sothat the antisense strand is transcribed. The term also refers to theantisense strand of RNA or of cDNA which compliments the strand of DNAencoding the protein or peptide of interest. In either case, whenintroduced into a cell under the control of a promoter, the anti-sensenucleic acid sequence inhibits the synthesis of the protein of interestfrom the endogenous gene. The inhibition appears to depend on theformation of an RNA-RNA or cDNA-RNA duplex in the nucleus or in thecytoplasm. Thus, if the antisense gene is stably introduced into acultured cell, the normal processing and/or transport is affected if asense-antisense duplex forms in the nucleus; or if antisense RNA isintroduced into the cytoplasm of the cell, the expression or translationof the endogenous product is inhibited.

“Antisense” refers particularly to the nucleic acid sequence of thenon-coding strand of a double stranded DNA molecule encoding a protein,or to a sequence which is substantially homologous to the non-codingstrand. As defined herein, an antisense sequence is complementary to thesequence of a double stranded DNA molecule encoding a protein. It is notnecessary that the antisense sequence be complementary solely to thecoding portion of the coding strand of the DNA molecule. The antisensesequence may be complementary to regulatory sequences specified on thecoding strand of a DNA molecule encoding a protein, which regulatorysequences control expression of the coding sequences.

Antisense nucleic acid sequences can further include modifications whichcan affect the biological activity of the antisense molecule, or itsmanner or rate of expression. Such modifications can also include, e.g.,mutations, insertions, deletions, or substitutions of one or morenucleotides that do not affect the function of the antisense molecule,but which may affect intracellular localization. Modifications include,but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxymethyl uracil, 5-carboxyhydroxymethyl-2-thiouridine,5-carboxymethylaminomethyl uracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentyladenine,1-methylguanine, 1-methylinosine, 2,2 dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methylaminomethyl-2-thioracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methyluracil,2-methylthio-N6-isopentenyladenine, uracil-5 oxyacetic acid,wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,5-methy-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, and2,6-diaminopurine.

The antisense nucleic acid sequence can determine an uninterruptedantisense RNA sequence or it can include one or more introns. Theantisense Jagged molecule(s) of the present invention are referred to as“γ-Jagged.”

The terms “complementary” and “antisense” as used herein, are notentirely synonymous. “Antisense” refers particularly to the nucleic acidsequence of the non-coding strand of a double stranded DNA moleculeencoding a protein, or to a sequence which is substantially homologousto the non-coding strand. “Complementary” as used herein refers to thebroad concept of subunit sequence complementarity between two nucleicacids, e.g., two DNA molecules. When a nucleotide position in both ofthe molecules is occupied by nucleotides normally capable of basepairing with each other, then the nucleic acids are considered to becomplementary to each other at this position. Thus, two nucleic acidsare complementary to each other when a substantial number (at least 50%)of corresponding positions in each of the molecules are occupied bynucleotides which normally base pair with each other (e.g., A:T and G:Cnucleotide pairs). As defined herein, an antisense sequence iscomplementary to the sequence of a double stranded DNA molecule encodinga protein. It is not necessary that the antisense sequence becomplementary solely to the coding portion of the coding strand of theDNA molecule. The antisense sequence may be complementary to regulatorysequences specified on the coding strand of a DNA molecule encoding aprotein, which regulatory sequences control expression of the codingsequences.

A “coding region” of a gene consists of the nucleotide residues of thecoding strand of the gene and the nucleotides of the non-coding strandof the gene which are homologous with or complementary to, respectively,the coding region of an mRNA molecule which is produced by transcriptionof the gene.

A “coding region” of an mRNA molecule also consists of the nucleotideresidues of the mRNA molecule which are matched with an anticodon regionof a transfer RNA molecule during translation of the mRNA molecule orwhich encode a stop codon. The coding region may thus include nucleotideresidues corresponding to amino acid residues which are not present inthe mature protein encoded by the mRNA molecule (e.g., amino acidresidues in a protein export signal sequence).

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(ie., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

The use of the term “DNA encoding” should be construed to include theDNA sequence which encodes the desired protein and any necessary 5′ or3′ untranslated regions accompanying the actual coding sequence.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

A “differentiation effective amount,” as the term is used herein, meansan amount of soluble Jagged that mediates a detectable increase ordecrease in the level of behavior associated with endothelial celldifferentiation. One skilled in the art would appreciate, based upon thedisclosure provided herein, that such amount depends on the nature ofthe cell, tissue, or organism to which the soluble Jagged isadministered. The skilled artisan would further appreciate, based uponthe disclosure provided herein, that there are a number of assays,several of which are disclosed elsewhere herein, useful for assessingthe level of differentiation, such as a modified differential displaymethod, endothelial cell (e.g., HUVEC) organization, endothelial cellmigration, sprout formation, as well as assays to be developed in thefuture, contemplated in the present invention.

By the term “expression inhibiting amount”, as the term is used herein,is meant an amount of soluble Jagged that mediates a detectable decreasein the level of type I collagen expression in a cell when the level oftype I collagen expression in the cell is compared to the level of typeI collagen expression in the same cell prior to administration ofsoluble Jagged or to the level of type I collagen in an otherwiseidentical cell to which soluble Jagged is not administered.

One skilled in the art would appreciate, based upon the disclosureprovided herein, that such amount depends on the nature of the cell ortissue from which the cell is obtained, and the amount of endogenoustype I collagen expression in the cell prior to or in the absence ofadministration of soluble Jagged.

The skilled artisan would further appreciate, based upon the disclosureprovided herein, that there are a number of assays, several of which aredisclosed elsewhere herein, useful for assessing the level of type Icollagen expression in a cell such as a differential display method(e.g., SAGE analysis), antibody-based detection of type I collagen genetranslation product in a cell (e.g., immunoblotting, ELISA,immunoprecipitation, and the like), and detection of nucleic acidencoding type I collagen (e.g., Southern blotting, Northern blotting,PCR-based assays, and the like), as well as assays to be developed inthe future.

A first region of an oligonucleotide “flanks” a second region of theoligonucleotide if the two regions are adjacent to one another or if thetwo regions are separated by no more than about 1000 nucleotideresidues, and preferably no more than about 100 nucleotide residues.

By the term “DNA segment” is meant a molecule comprising a linearstretch of nucleotides wherein the nucleotides are present in a sequencethat encodes, through the genetic code, a molecule comprising a linearsequence of amino acid residues that is referred to as a protein, aprotein fragment, or a polypeptide.

“Gene,” as used herein, refers to a single polypeptide chain or protein,and as used herein includes the 5′ and 3′ ends. The polypeptide can beencoded by a full-length sequence or any portion of the coding sequence,so long as the functional activity of the protein is retained.

A “complementary DNA” or “cDNA” gene includes recombinant genessynthesized by reverse transcription of messenger RNA (“mRNA”) lackingintervening sequences (introns).

“Structural gene” means a DNA sequence that is transcribed into mRNAthat is then translated into a sequence of amino acids characteristic ofa specific polypeptide. According to art-recognized convention, thefirst nucleotide of the first translated codon is numbered +1, and thenucleotides are numbered consecutively with positive integers throughthe translated region of the structural gene and into the 3′untranslated region. The numbering of the nucleotides in the promoterand/or regulatory region 5′ to the translated region proceedsconsecutively with negative integers with the 5′ nucleotide next to thefirst translated nucleotide being numbered −1.

By the term “gel electrophoresis,” is meant assay to assess the size ofparticular DNA fragments. More specifically, the most common technique(although not the only one) to determine the size of a nucleic acidfragment, is agarose gel electrophoresis, which is based on theprinciple that DNA molecules migrate through the gel as though it were asieve that retards the movement of the largest molecules to the greatestextent, and the movement of the smallest molecules to the least extent.The fractionated molecules can be visualized by staining, permitting theDNA fragments of a genome to be visualized. Such techniques arewell-known in the art and the gel matrix can be comprised of a varietyof substances including, but not limited to, agarose, acrylamide, andthe like, as described in, e.g., Sambrook et al. (1989, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NewYork), Ausubel et al. (1997, Current Protocols in Molecular Biology,Green & Wiley, New York), and other standard treatises.

Most genomes, including the human genome, contain too many DNA sequencesto produce an easily visualized pattern. Thus, a methodology referred as“Southern hybridization” (or “blotting”) is used to visualize smallsubsets of fragments. By this procedure the fractionated DNA isphysically transferred onto nitrocellulose filter paper or anotherappropriate surface using recognized methods. Note that RNA fragmentscan be similarly visualized by the “northern blot” process.

By the term “nucleic acid hybridization,” is meant a process by whichtwo single-stranded nucleic acid molecules will bind with each other.The process depends on the principle that two single-stranded moleculesthat have complementary base sequences will reform into thethermodynamically favored double-stranded configuration (“reanneal”) ifthey are mixed in solution under the proper conditions. The reanneallingprocess can occur even if one of the single strands is immobilized.

“Homologous” as used herein, refers to the subunit sequence similaritybetween two polymeric molecules, e.g., between two nucleic acidmolecules, e.g., two DNA molecules or two RNA molecules, or between twopolypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit, e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions, e.g.,if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two compound sequences are homologous then the twosequences are 50% homologous, if 90% of the positions, e.g., 9 of 10,are matched or homologous, the two sequences share 90% homology. By wayof example, the DNA sequences 3′ATTGCC5′ and 3′ TATGGC share 50%homology.

As used herein, “homology” is used synonymously with “identity.”

In addition, when the term “homology” is used herein to refer to thenucleic acids and proteins, it should be construed to be applied tohomology at both the nucleic acid and the amino acid levels.

A first oligonucleotide anneals with a second oligonucleotide with “highstringency” if the two oligonucleotides anneal under conditions wherebyonly oligonucleotides which are at least about 60%, more preferably atleast about 65%, even more preferably at least about 70%, yet morepreferably at least about 80%, and preferably at least about 90% or,more preferably, at least about 95% complementary anneal with oneanother. The stringency of conditions used to anneal twooligonucleotides is a function of, among other factors, temperature,ionic strength of the annealing medium, the incubation period, thelength of the oligonucleotides, the G-C content of the oligonucleotides,and the expected degree of non-homology between the twooligonucleotides, if known. Methods of adjusting the stringency ofannealing conditions are known (see, e.g., Sambrook et al., 1989,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York).

The determination of percent identity between two nucleotide or aminoacid sequences can be accomplished using a mathematical algorithm. Forexample, a mathematical algorithm useful for comparing two sequences isthe algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA87:2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl.Acad. Sci. USA 90:5873-5877). This algorithm is incorporated into theNBLAST and XBLAST programs of Altschul, et al. (1990, J. Mol. Biol.215:403-410), and can be accessed, for example, at the National Centerfor Biotechnology Information (NCBI) world wide web site. BLASTnucleotide searches can be performed with the NBLAST program (designated“blastn” at the NCBI web site), using the following parameters: gappenalty=5; gap extension penalty=2; mismatch penalty=3; match reward=1;expectation value 10.0; and word size=11 to obtain nucleotide sequenceshomologous to a nucleic acid described herein. BLAST protein searchescan be performed with the XBLAST program (designated “blastn” at theNCBI web site) or the NCBI “blastp” program, using the followingparameters: expectation value 10.0, BLOSUM62 scoring matrix to obtainamino acid sequences homologous to a protein molecule described herein.

To obtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al. (1997, Nucleic Acids Res.25:3389-3402). Alternatively, PSI-Blast or PHI-Blast can be used toperform an iterated search which detects distant relationships betweenmolecules (id.) and relationships between molecules which share a commonpattern. When utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blastprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See National Center for BiotechnologyInformation world wide web site.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically exact matches arecounted.

As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame encoding a polypeptideof the invention. Such natural allelic variations can typically resultin 1-5% variance in the nucleotide sequence of a given gene. Alternativealleles can be identified by sequencing the gene of interest in a numberof different individuals. This can be readily carried out by usinghybridization probes to identify the same genetic locus in a variety ofindividuals. Any and all such nucleotide variations and resulting aminoacid polymorphisms or variations that are the result of natural allelicvariation and that do not alter the functional activity are intended tobe within the scope of the invention.

Moreover, nucleic acid molecules encoding proteins of the invention fromother species (homologs), which have a nucleotide sequence which differsfrom that of the human proteins described herein are within the scope ofthe invention. Nucleic acid molecules corresponding to natural allelicvariants and homologs of a cDNA of the invention can be isolated basedon their identity to human nucleic acid molecules using the human cDNAs,or a portion thereof, as a hybridization probe according to standardhybridization techniques under stringent hybridization conditions. Forexample, a cDNA encoding a soluble form of a membrane-bound protein ofthe invention, Jagged-1, can be isolated based on its hybridization witha nucleic acid molecule encoding all or part of the membrane-bound form.Likewise, a cDNA encoding a membrane-bound form can be isolated based onits hybridization with a nucleic acid molecule encoding all or part ofthe soluble form.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, e.g., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, e.g., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, e.g., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytidine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the composition of the inventionfor its designated use. The instructional material of the kit of theinvention may, for example, be affixed to a container which contains thecomposition or be shipped together with a container which contains thecomposition. Alternatively, the instructional material may be shippedseparately from the container with the intention that the instructionalmaterial and the composition be used cooperatively by the recipient.

The term “ligand,” as used herein, refers to any protein or proteinsthat can interact with a receptor binding domain, thus having a “bindingaffinity” for such domain. Ligands can be soluble or membrane bound, andthey can be a naturally occurring protein, or synthetically orrecombinantly produced. The ligand can also be a nonprotein moleculethat acts as ligand when it interacts with the receptor binding domain.Interactions between the ligand and receptor binding domain include, butare not limited to, any covalent or non-covalent interactions. Thereceptor binding domain is any region of the receptor molecule, e.g.,Notch, that interacts directly or indirectly with the ligand, e.g.,Jagged. If the Notch-Jagged interaction acts as an on-off switch, Jaggedcan provide the receptor binding domain, and Notch or a componentproduced as a result of the Notch-Jagged interaction can act as theligand.

“Mutants,” “derivatives,” and “variants” of the peptides of theinvention (or of the DNA encoding the same) are peptides which may bealtered in one or more amino acids (or in one or more base pairs) suchthat the peptide (or nucleic acid) is not identical to the sequencesrecited herein, but has the same property as the soluble Jagged peptidesdisclosed herein, in that the peptide has the property of inducingexpression of certain genes as assessed using SAGE analysis (e.g.,enhancer of split groucho, type IV collagenase, connexin 32, cathepsinD, and vimentin; mediating reduced level of expression of certain genes(e.g., pro-α-2(I) collagen, FGFR-1, and IkB-β), affecting endothelialsprout formation, affecting angiogenesis, the ability to developangiogenic tissue masses in nude mice, the ability to induceangiogenesis in a CAM angiogenesis model, and the like.

A “functional derivative” of a sequence, either protein or nucleic acid,is a molecule that possesses a biological activity (either functional orstructural) that is substantially similar to a biological activity ofJagged protein or a nucleic acid sequence encoding Jagged, or a portionthereof. A functional derivative of a protein may or may not containpost-translational modifications such as covalently linked carbohydrate,depending on the necessity of such modifications for the performance ofa specific function. The term “functional derivative” is intended toinclude the “fragments,” “segments,” “variants,” “analogs,” or “chemicalderivatives” of a molecule.

As used herein, a molecule is said to be a “chemical derivative” ofanother molecule when it contains additional chemical moieties notnormally a part of the molecule. Such moieties can improve themolecule's solubility, absorption, biological half life, and the like.The moieties can alternatively decrease the toxicity of the molecule,eliminate or attenuate any undesirable side effect of the molecule, andthe like. Moieties capable of mediating such effects are disclosed in,for example, Remington's Pharmaceutical Sciences (1980, Mack PublishingCo., Easton, Pa.). Procedures for coupling such moieties to a moleculeare well known in the art.

A “variant” or “allelic or species variant” of a protein or nucleic acidis meant to refer to a molecule substantially similar in structure andbiological activity to either the protein or nucleic acid. Thus,provided that two molecules possess a common activity and may substitutefor each other, they are considered variants as that term is used hereineven if the composition or secondary, tertiary, or quaternary structureof one of the molecules is not identical to that found in the other, orif the amino acid or nucleotide sequence is not identical.

By describing two polynucleotides as “operably linked” is meant that asingle-stranded or double-stranded nucleic acid moiety comprises the twopolynucleotides arranged within the nucleic acid moiety in such a mannerthat at least one of the two polynucleotides is able to exert aphysiological effect by which it is characterized upon the other. By wayof example, a promoter operably linked to the coding region of a gene isable to promote transcription of the coding region.

Preferably, when the nucleic acid encoding the desired protein furthercomprises a promoter/regulatory sequence, the promoter/regulatorysequence is positioned at the 5′ end of the desired protein codingsequence such that it drives expression of the desired protein in acell.

As used herein, the term “pharmaceutically acceptable carrier” means achemical composition with which the active ingredient may be combinedand which, following the combination, can be used to administer theactive ingredient to a subject.

As used herein, the term “physiologically acceptable” ester or saltmeans an ester or salt form of the active ingredient which is compatiblewith any other ingredients of the pharmaceutical composition, which isnot deleterious to the subject to which the composition is to beadministered.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell under most or allphysiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell substantially only whenan inducer which corresponds to the promoter is present in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a cell substantiallyonly if the cell is a cell of the tissue type corresponding to thepromoter.

By the term “exogenous nucleic acid” is meant that the nucleic acid hasbeen introduced into a cell or an animal using technology which has beendeveloped for the purpose of facilitating the introduction of a nucleicacid into a cell or an animal.

The term “expression of a nucleic acid “as used herein means thesynthesis of the protein product encoded by the nucleic acid. Morespecifically, expression is the process by which a structural geneproduces a polypeptide. It involves transcription of the gene into mRNA,and the translation of such mRNA into a polypeptide.

By the term “positioned at the 5′ end” as used herein, is meant that thepromoter/regulatory sequence is covalently bound to the 5′ end of thenucleic acid whose expression it regulates, at a position sufficientlyclose to the 5′ start site of transcription of the nucleic acid so as todrive expression thereof.

The direction of 5′ to 3′ addition of nucleotides to nascent RNAtranscripts is referred to as the transcription direction. The DNAstrand having the same sequence as an mRNA is referred to as the “codingstrand”; sequences on the DNA strand which are located 5′ to a referencepoint on the DNA are referred to as “upstream sequences”; sequences onthe DNA strand which are 3′ to a reference point on the DNA are referredto as “downstream sequences.”

A “portion” of a polynucleotide means at least about twenty sequentialnucleotide residues of the polynucleotide. It is understood that aportion of a polynucleotide may include every nucleotide residue of thepolynucleotide.

A “polyadenylation sequence” is a polynucleotide sequence which directsthe addition of a poly A tail onto a transcribed messenger RNA sequence.

A “polynucleotide” means a single strand or parallel and anti-parallelstrands of a nucleic acid. Thus, a polynucleotide may be either asingle-stranded or a double-stranded nucleic acid.

The term “nucleic acid” typically refers to large polynucleotides.

The term “oligonucleotide” typically refers to short polynucleotides,generally, no greater than about 50 nucleotides. It will be understoodthat when a nucleotide sequence is represented by a DNA sequence (i.e.,A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) inwhich “U” replaces “T.”

The term “oligonucleotide or oligomer”, as used herein, refers to amolecule comprised of two or more deoxyribonucleotides orribonucleotides, preferably more than three. Its exact size will dependon many factors, which in turn depend on the ultimate function or use ofthe oligonucleotide. An oligonucleotide may be derived synthetically orby cloning.

Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction.

“Primer” refers to a polynucleotide that is capable of specificallyhybridizing to a designated polynucleotide template and providing apoint of initiation for synthesis of a complementary polynucleotide.Such synthesis occurs when the polynucleotide primer is placed underconditions in which synthesis is induced, i.e., in the presence ofnucleotides, a complementary polynucleotide template, and an agent forpolymerization such as DNA polymerase. A primer is typicallysingle-stranded, but may be double-stranded. Primers are typicallydeoxyribonucleic acids, but a wide variety of synthetic and naturallyoccurring primers are useful for many applications. A primer iscomplementary to the template to which it is designed to hybridize toserve as a site for the initiation of synthesis, but need not reflectthe exact sequence of the template. In such a case, specifichybridization of the primer to the template depends on the stringency ofthe hybridization conditions. Primers can be labeled with, e.g.,chromogenic, radioactive, or fluorescent moieties and used as detectablemoieties.

By the term “amplification primer”, as used herein, is meant anoligonucleotide which is capable of annealing adjacent to a targetsequence and serving as an initiation point for DNA synthesis whenplaced under conditions in which synthesis of a primer extension productwhich is complementary to a nucleic acid strand is initiated.

“Probe” refers to a polynucleotide that is capable of specificallyhybridizing to a designated sequence of another polynucleotide. A probespecifically hybridizes to a target complementary polynucleotide, butneed not reflect the exact complementary sequence of the template. Insuch a case, specific hybridization of the probe to the target dependson the stringency of the hybridization conditions. Probes can be labeledwith, e.g., chromogenic, radioactive, or fluorescent moieties and usedas detectable moieties.

One skilled in the art would appreciate, based upon the disclosureprovided herein, that to visualize a particular DNA sequence in ahybridization procedure, a labeled DNA molecule or “hybridization probe”can be reacted to a fractionated nucleic acid bound to a nitrocellulosefilter. The areas on the filter that carry nucleic acid sequencescomplementary to the labeled DNA probe become labeled themselves as aconsequence of the reannealing reaction. The areas of the filter thatexhibit such labeling are visualized. The hybridization probe isgenerally produced by molecular cloning of a specific DNA sequence.

“Recombinant polynucleotide” refers to a polynucleotide having sequencesthat are not naturally joined together. An amplified or assembledrecombinant polynucleotide may be included in a suitable vector, and thevector can be used to transform a suitable host cell.

A recombinant polynucleotide may serve a non-coding function (e.g.,promoter, origin of replication, ribosome-binding site, etc.) as well.

A “recombinant polypeptide” is one which is produced upon expression ofa recombinant polynucleotide.

“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. Synthetic polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.

The term “protein” typically refers to large polypeptides.

The term “peptide” typically refers to short polypeptides.

Conventional notation is used herein to portray polypeptide sequences:the left-hand end of a polypeptide sequence is the amino-terminus; theright-hand end of a polypeptide sequence is the carboxyl-terminus.

A cell that comprises an exogenous nucleic acid is referred to as a“recombinant cell.” Such a cell may be a eukaryotic cell or aprokaryotic cell. A gene which is expressed in a recombinant cellwherein the gene comprises a recombinant polynucleotide, produces a“recombinant polypeptide.”

“Sequence amplification,” as the term is used herein, means a method forgenerating large amounts of a target sequence. In general, one or moreamplification primers are annealed to a nucleic acid sequence. Usingappropriate enzymes, sequences found adjacent to, or in between theprimers are amplified.

By the term “specifically binds,” as used herein, is meant a compound,e.g., a protein, a nucleic acid, an antibody, and the like, whichrecognizes and binds a specific molecule, but does not substantiallyrecognize or bind other molecules in a sample.

“Steady-state level” refers to a stable condition that does not changeover time, or the state in which change in one direction or productionof a component is continually balanced by a compensatory change inanother.

A “substantially pure” protein or nucleic acid is a protein or nucleicacid preparation that is generally lacking in other cellular componentswith which it is normally associated in vivo. That is, as used herein,the term “substantially pure” describes a compound, e.g., a nucleicacid, protein or polypeptide, which has been separated from componentswhich naturally accompany it. Typically, a compound is substantiallypure when at least about 10%, preferably at least about 20%, morepreferably at least about 50%, still more preferably at least about 75%,even more preferably at least about 90%, and most preferably at leastabout 99% of the total material (by volume, by wet or dry weight, or bymole percent or mole fraction) in a sample is the compound of interest.Purity can be measured by any appropriate method, e.g., by columnchromatography, gel electrophoresis or HPLC analysis.

A compound, e.g., a nucleic acid, a protein or polypeptide is also“substantially purified” when it is essentially free of naturallyassociated components or when it is separated from the nativecontaminants which accompany it in its natural state. Thus, a“substantially pure” preparation of a nucleic acid, as used herein,refers to a nucleic acid sequence which has been purified from thesequences which flank it in a naturally occurring state, e.g., a DNAfragment which has been removed from the sequences which are normallyadjacent to the fragment in a genome in which it naturally occurs.

Similarly, a “substantially pure” preparation of a protein or apolypeptide, as used herein, refers to a protein or polypeptide whichhas been purified from components with which it is normally associatedin its naturally occurring state. A substantially pure peptide can bepurified by following known procedures for protein purification, whereinan immunological, enzymatic or other assay is used to monitorpurification at each stage in the procedure. Protein purificationmethods are well known in the art, and are described, for example inDeutscher et al. (1990, In: Guide to Protein Purification, HarcourtBrace Jovanovich, San Diego).

By “tag” polypeptide is meant any protein which, when linked by apeptide bond to a protein of interest, may be used to localize theprotein, to purify it from a cell extract, to immobilize it for use inbinding assays, or to otherwise study its biological properties and/orfunction. A chimeric (i.e., fusion) protein containing a “tag” epitopecan be immobilized on a resin which binds the tag. Such tag epitopes andresins which specifically bind them are well known in the art andinclude, for example, tag epitopes comprising a plurality of sequentialhistidine residues (His6), which allows isolation of a chimeric proteincomprising such an epitope on nickel-nitrilotriacetic acid-agarose, ahemagglutinin (HA) tag epitope allowing a chimeric protein comprisingsuch an epitope to bind with an anti-HA-monoclonal antibody affinitymatrix, a myc tag epitope allowing a chimeric protein comprising such anepitope to bind with an anti-myc-monoclonal antibody affinity matrix, aglutathione-S-transferase tag epitope, and a maltose binding protein(MBP) tag epitope, which can induce binding between a protein comprisingsuch an epitope and a glutathione- or maltose-Sepharose column,respectively. Production of proteins comprising such tag epitopes iswell known in the art and is described in standard treatises such asSambrook et al., 1989, supra, and Ausubel et al., supra. Likewise,antibodies to the tag epitope (e.g., anti-HA, anti-myc antibody 9E10,and the like) allow detection and localization of the fusion protein in,for example, Western blots, ELISA assays, and immunostaining of cells.

By the term “type I collagen,” as used herein, is meant any collagenknown to be a type I collagen, e.g., pro-α1(I) collagen, pro-α2(I)collagen, and the like, as well as other collagens identified as type Icollagen in the future according to criteria that are well-known in theart.

A “vector,” as used herein, refers to a plasmid or phage DNA or otherDNA sequence into which DNA may be inserted to be cloned. The vector canreplicate autonomously in a host cell, and can be further characterizedby one or a small number of endonuclease recognition sites at which suchDNA sequences can be cut in a determinable fashion and into which DNAmay be inserted. The vector can further contain a marker suitable foruse in the identification of cells transformed with the vector. Thewords “cloning vehicle” are sometimes used for “vector.”

Additionally, the term “vector” encompasses any plasmid, phage and virusencoding an exogenous nucleic acid. The term also includes non-plasmidand non-viral compounds which facilitate transfer of nucleic acid intovirions or cells, such as, for example, polylysine compounds and thelike. The vector can be a viral vector which is suitable as a deliveryvehicle for delivery of the nucleic acid encoding, e.g., Jagged, solubleJagged, γ-Jaggged, and/or or a portion thereof, to a cell and/or apatient, or the vector can be a non-viral vector which is suitable forthe same purpose.

Examples of viral and non-viral vectors for delivery of DNA to cells andtissues are well-known in the art and are described, for example, in Maet al. (1997, Proc. Natl. Acad. Sci. U.S.A. 94:12744-12746). Examples ofviral vectors include, but are not limited to, a recombinant vacciniavirus, a recombinant adenovirus, a recombinant retrovirus, a recombinantadeno-associated virus, a recombinant avian pox virus, and the like(Cranage et al., 1986, EMBO J. 5:3057-3063; International PatentApplication No. WO94/17810, published Aug. 18, 1994; InternationalPatent Application No. WO94/23744, published Oct. 27, 1994). Examples ofnon-viral vectors include, but are not limited to, liposomes, polyaminederivatives of DNA, and the like.

“Expression vector,” as the term is used herein, means a vector orvehicle similar to a cloning vector but which is capable of expressing agene which has been cloned into it, after transformation into a host.The cloned gene is usually placed under the control of (i.e., operablylinked thereto) certain regulatory/control sequences such as, e.g.,promoter sequences. Expression control sequences will vary depending onwhether the vector is designed to express the operably linked gene in aprokaryotic or eukaryotic host and can additionally containtranscriptional elements such as enhancer elements, terminationsequences, tissue-specificity elements, and/or translational initiationand termination sites. One skilled in the art would appreciate, basedupon the disclosure provided herein and methods well-known in the art,that not all regulatory/control elements need be present in allconstructs; rather, the present invention encompasses an expressionvector comprising any combination of elements known in the art such thata nucleic acid of interest is expressed as desired.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., retroviruses, adenoviruses, and adeno-associatedviruses) that incorporate the recombinant polynucleotide.

Description

Angiogenesis, or the formation of new blood vessels, plays a centralrole in a number of physiologic and pathologic conditions, includingplacental development, wound healing, rheumatoid arthritis, diabeticretinopathy and solid tumor growth and metastasis. Endothelial cellscomprise a monolayer lining the luminal surface of all blood vessels,thereby playing a central role in this process. In vitro populations ofendothelial cells isolated from both large vessels and microvessels canbe induced to mimic this differentiation process by forming acapillary-like network. Three-dimensional fibrin gels have been used tomimic angiogenesis, as an in vitro corollary of the in vivo phenomenonsince endothelial cells invade blood clots in the process of woundrepair.

Cellular differentiation is a well documented process in vitro,generally requiring a transcriptional component for induction. However,in contrast to most cell types, endothelial cell differentiation hasbeen shown to be reversible. Digestion of the endothelial cellularnetworks formed in vitro, and subsequent culture of the cells in thepresence of FGF-1 causes them to revert to an undifferentiated phenotype(see, e.g., Maciag et al., 1982, J. Cell Biol. 94:511-520). However,endothelial cell differentiation has also been shown to have atranscriptional basis. Endothelial cell (HUVEC) organization into acellular network has been shown to be associated with an increase in thetranscript encoding fibronectin, and a decrease in the transcriptencoding sis, which reverses when the cellular network is digested withproteases and the cells revert to the proliferative phenotype (see,e.g., Jaye et al., 1985, Science 228:882-885).

HUVEC are capable of two different behaviors, both of which are termed“differentiation.” The first is the formation of a two dimensionalnetwork involving cell elongation, anastomosis and branching that doesnot require transcription and translation, but requirespost-translational modification. The second is a more complexthree-dimensional process resulting in a capillary network containinglumens, which Zimrin et al. (1995), have shown requires bothtranscriptional and post-translational events. In addition, Zimrin etal. (1995), has defined the modified differential display technique asapplied to endothelial cells and demonstrated that it is a very usefulmethod of isolating transcripts which are differentially expressed asendothelial cells differentiate.

Thus, in the present invention, using a modification of the differentialdisplay method, the human homolog of the Jagged ligand for the Notchreceptor has been isolated from human umbilical vein endothelial cells(HUVEC) invading a fibrin gel. The addition of an antisense Jaggedoligonucleotide to bovine microvascular endothelial cells on collagenresulted in a marked increase in their invasion into the collagen gel inresponse to FGF-2. However, while the antisense Jagged oligonucleotideof the present invention was also able to increase the migration ofbovine microvascular endothelial cells on fibronectin, theoligonucleotide significantly decreased the migration of bovineendothelial cells derived from the aorta, suggesting a divergence in themechanism utilized by two different endothelial cell populations torespond to the Notch signaling system.

The distinction between microvascular and large vessel endothelium iswell recognized as a part of the heterogeneity of the vascularendothelium in general and this is reflected in the properties ofendothelial cells from different sources in cell culture (Carson andHaudenschild, 1986, In Vitro 22:344-354), and in organ-specificexpression of different adhesion molecules, cell surface glycoproteinsand lectin-binding sites (Gumkowski et al., 1987, Blood Vessels 24:11).

Briefly, to identify the molecular events necessary in the process ofangiogenesis, a modified differential display procedure was used toisolate messages that were differentially expressed in HUVEC plated onfibrin in the presence of FGF-1 over the course of 24 hours. Asdescribed in Example 2, infra, one of the cDNAs that was amplified at 2hours, and which was found to be highly homologous to the rat Jaggedtranscript was identified as an isolate of the human Jagged homolog. Theputative protein sequence of the present invention includes a signalpeptide, a DSL domain shared by the Notch ligands Delta, Serrate, Lag-2and Apx-1, sixteen tandem epidermal growth factor-like repeats, acysteine-rich region, a transmembrane domain and a cytoplasmic tail. The5′ end of the sequence of the human Jagged isolate corresponds toposition 417 of the rat sequence, at the eleventh codon of the predicted21 residue signal peptide.

To investigate the role of Jagged and Notch in endothelial cellbehavior, reverse transcription and polymerase chain reactionamplification (RT-PCR) was used to evaluate the steady-state messagelevels of Jagged and two related Notch proteins, human TAN-1 and humanNotch group protein, in human endothelial cells on fibrin (FIG. 5).Although the Jagged message was found to be up-regulated in populationsof HUVEC exposed to fibrin at the 3 hour timepoint, the message levelsof the two Notch proteins was not changed over the course of 24 hours.Thus, it is shown in the present invention that the human endothelialcell population is capable of expressing both the Jagged ligand and theNotch receptor, indicating that the human endothelial cell is completingan autocrine signal using the Notch signal transduction pathway. Thedata do not distinguish, however, between a homogeneous populationexpressing both Notch and Jagged proteins, or heterogeneoussubpopulations of endothelial cells that display Notch, Jagged, both orneither protein.

Therefore, to delineate a functional role for Jagged, an antisenseJagged oligonucleotide was designed in the present invention, whichencompassed the Kozak consensus region, the ATG start codon and the nextthree codons of the rat Jagged cDNA sequence. Similar strategies havepreviously proved useful as a means of repressing the translationalefficiency of a wide variety of transcripts in vitro (see Scanlon etal., 1995, FASEB J. 9:1288-1296; Maier et al., 1990, J. Biol. Chem.265:10805-10808).

Because endothelial cell migration is an important component ofangiogenesis, endothelial cell behavior was evaluated under conditionsof sprout formation (Montesano and Orci, 1985, Cell 42:469-477) andmigration (Sato and Rifkin, 1988, J. Cell Biol. 107:1199-1205). Theaddition of the oligonucleotide to bovine microvascular endothelialcells plated on collagen at varying concentrations resulted in anoligonucleotide-induced dose-dependent increase in the total length ofsprout formation observed in response to the addition of FGF-2 (FIG. 6).The addition of several control oligonucleotides, including a senseoligonucleotide covering the same sequence, a 5′ antisenseoligonucleotide with every third base mutated, and a randomoligonucleotide, had no effect on the total length of sprout formation(FIG. 6). Thus, the addition of the antisense Jagged oligonucleotidesignificantly enhanced endothelial cell sprout formation beyond thelevel achieved by FGF-2.

These data were unusual since endothelial cell sprout formation requirescell migration as a component, and the Jagged cDNA had been isolatedfrom a human endothelial cell system where migration into the fibrinclot also occurs. Consequently, the effect of the antisense Jaggedoligonucleotide was studied on capillary and large vessel endothelialcell migration, respectively. It was found that while a bovinemicrovascular endothelial cell population exhibited a significantdose-dependent increase in their migration in the presence of the Jaggedantisense oligonucleotide (FIG. 7A), the migration of bovine aortaendothelial cells was significantly attenuated in a dose-dependentfashion by the antisense Jagged oligonucleotide (FIG. 7B). Thus, theability of Jagged-Notch signaling to modify endothelial cells wasdependent upon the anatomic source of the endothelial cells.

Since the endothelial cells studied were from both large and smallvessels responded to the antisense Jagged oligonucleotide in a disparatemanner, and both cellular populations are likely to express the Notchreceptor, the difference in their response to the Jagged antisenseoligonucleotide indicates for the first time that there are differencesbetween large and small vessels in the Notch signaling pathway. Althoughit has been documented that cells isolated from small vessels are ableto undergo the phenotypic changes characteristic of capillary formationmore readily than endothelial cells isolated from large vessels (Ingberand Folkman, 1989, J. Cell Biol. 109:317-330), the novel response to theJagged antisense oligonucleotide disclosed in the present inventionrepresents the first demonstration of an effect not only different indegree but also in direction.

The present embodiments further demonstrate that the addition ofexogenous Jagged (or enhanced expression of Jagged) produces an effectopposite to that seen in Examples 5-7. In other words, the addition orincreased expression of Jagged decreases the migration and invasion ofmicrovascular cells from the vaso vasorum, and increases or stimulatesthe migration of large vessel endothelial cells.

The clinical importance of the disparate effect of the Jagged-Notchsignaling pathway on the macro- and micro-diameter blood vessels issignificant, offering a solution to many aspects of vascularpathophysiology. For example, the morbidity and mortality fromhypertension is clearly based on the disease of the large vessels(atherosclerosis and stroke), but in the major forms of hypertension,the actual cause for elevated blood pressure lies in the peripheralvascular beds (arterioles and microvasculature) (Chobanian et al., 1986,Hypertension 8:15-21). The presently defined compositions and methodsmay resolve the previously unanswered question of how hypertension couldbe directly related to the aortic intima and atherosclerosis, and viceversa, how known atherogenic risk factors could affect the microvascularendothelium (Chan et al., 1979, Microvasc. Res. 18:353-369).

Moreover, the presently embodied compositions and methods are useful forthe modification of a post-angioplastic situation, when one or morelarge coronary vessel have been stripped of their endothelial celllining. One of the most serious complications limiting the value of theangioplastic procedure is the occurrence of restenosis or the rapidmigration and proliferation of smooth muscle cells,monocytes/macrophages, platelets, and endothelium at the wound siteresulting in a reocclusion of the vessel that may be more extensive thanbefore treatment (see numerous review articles on the subject, e.g.,Schwartz et al., 1981, Atherosclerosis 1:107-161). However, treating thewounded or injured area with a therapeutic amount of additionalrecombinant Jagged protein, or a functionally equivalent drug or proteinhaving the ability to signal Notch, will prevent or inhibit reocclusionby increasing the migration of the large vessel endothelial cells on theborders of the lesion into the denuded area to cover the lesion, whilealso decreasing emergence of the micro-vascular cells (smooth muscle,endothelial, macrophage, etc.) from the vaso vasorum and providing thenutrient microvessels or sprouts to supply the proliferating smoothmuscle cells.

In a preferred embodiment, the present invention provides highlypurified Jagged protein. As used herein, a protein is said to be highlypurified if the protein possesses a specific activity that is greaterthan that found in whole cell extracts containing the protein.

Any eukaryotic organism can be used as a source of Jagged, or the genesencoding the same, as long as the source organism naturally contains theligand or its equivalent. As used herein, “source organism” refers tothe original organism from which the amino acid or DNA sequence isderived, regardless of the organism the ligand is expressed in orultimately isolated from. For example, a human is said to be the “sourceorganism” of Jagged expressed by an insect expression system as long asthe amino acid sequence is that of human Jagged. The most preferredsource organism is human.

A variety of methodologies known in the art can be utilized to obtainthe Jagged proteins of the present invention. In one embodiment, theJagged is purified from tissues or cells which naturally produce it,such as HUVEC. One skilled in the art can readily follow known methodsfor isolating proteins in order to obtain the Jagged protein. Theseinclude, but are not limited to, immunochromatography, size-exclusionchromatography, ion-exchange chromatography, affinity chromatography,HPLC, and the methods set forth by example in the present disclosure.One skilled in the art can readily adapt known purification schemes todelete certain steps or to incorporate additional purificationprocedures.

In another embodiment, the ligand is purified from cells which have beenaltered to express the desired protein. As used herein, a cell is saidto be “altered to express a desired protein” when the cell, throughgenetic manipulation, is made to produce a protein which it normallydoes not produce, or which the cell normally produces at low levels. Oneskilled in the art can readily adapt procedures for introducing andexpressing either genomic or cDNA sequences into either eukaryotic orprokaryotic cells, in order to generate a cell which produces thedesired protein.

There are a variety of source organisms for DNA encoding the desiredprotein. The more preferred source is the endothelial cell. The mostpreferred source is the human endothelial cell. The embodied methods arereadily adapted to use of an HUVEC population as a model to be evaluatedin comparison with HU artery (A) EC and human cells obtained from otheranatomic sites. These include human adipose-derived microvascularendothelial cells (HMEC), human dermis-derived capillary endothelialcells (HCEC) and human saphenous vein (HSVEC) and artery (HSAEC). Manyhuman endothelial cell populations are readily available from commercial(HMEC and HCEC) and academic sources (HSVEC and HSAEC were provided byDr. Michael Watkins, Dept. of Surgery, Boston University; and HUAEC wereprovided by Dr. Victor van Hinsbergh, Gabius Institute, Netherlands).

In yet another embodiment, since probes are available which are capableof hybridizing to Jagged, DNA sequences encoding the desired nucleicacid sequence encoding the protein of interest can be obtained byroutine hybridization and selection from any host which possesses thesereceptors. A nucleic acid molecule, such as DNA, is said to be “capableof expressing” a polypeptide if it contains nucleotide sequences whichcontain transcriptional and translational regulatory information andsuch sequences are “operably linked” to nucleotide sequences whichencode the polypeptide. An operable linkage is a linkage in which theregulatory DNA sequences and the DNA sequence sought to be expressed areconnected in such a way as to permit gene sequence expression. Theprecise nature of the regulatory regions needed for gene sequenceexpression may vary from organism to organism, but shall in generalinclude a promoter region which, in prokaryotes, contains both thepromoter (which directs the initiation of RNA transcription) as well asthe DNA sequences which, when transcribed into RNA, will signal theinitiation of protein synthesis. Such regions will normally includethose 5′-non-coding sequences involved with initiation of transcriptionand translation, such as the TATA box, capping sequence, CAAT sequence,and the like.

If desired, the non-coding region 3′ to the gene sequence encodingJagged may be obtained by the above-described methods. This region maybe retained for its transcriptional termination regulatory sequences,such as termination and polyadenylation. Thus, by retaining the3′-region naturally contiguous to the DNA sequence encoding Jagged, thetranscriptional termination signals may be provided. Where thetranscriptional termination signals are not satisfactorily functional inthe expression host cell, then a 3′ region functional in the host cellmay be substituted.

Two DNA sequences (such as a promoter region sequence and the Jaggedencoding sequence) are said to be operably linked if the nature of thelinkage between the two DNA sequences does not (1) result in theintroduction of a frame-shift mutation, (2) interfere with the abilityof the promoter region sequence to direct the transcription of theJagged gene sequence, or (3) interfere with the ability of the Jaggedgene sequence to be transcribed by the promoter region sequence. Thus, apromoter region would be operably linked to a DNA sequence if thepromoter were capable of effecting transcription of that DNA sequence.To express Jagged, transcriptional and translational signals recognizedby an appropriate host are necessary.

In another embodiment, the nucleic acid sequences of the presentinvention are under controlled expression by the animal or humanpatient. In the alternative, the nucleic acids sequences areadministered to the patient in need of gene therapy, intravenously,intramuscularly, subcutaneously, enterally, topically, parenterally orsurgically. When administering the nucleic acids by injection, theadministration may be by continuous administration, or by single ormultiple administrations. The gene therapy is intended to be provided tothe recipient mammal in a “pharmacologically or pharmaceuticallyacceptable form” in an amount sufficient to be “therapeuticallyeffective.” The nucleic acid is said to be in “pharmaceutically orpharmacologically acceptable form” if its administration can betolerated by a recipient patient. An amount is said to be“therapeutically effective” (also referred to here and elsewhere as “aneffective amount”) if the dosage, route of administration, etc., of theagent are sufficient to affect a response to Jagged. The nucleic acid isconsidered to be in “pharmaceutically or pharmacologically acceptableform” if its administration can be tolerated by a recipient patient.

The present invention further encompasses the expression of the Jaggedprotein (or a functional derivative thereof) in either prokaryotic oreukaryotic cells. Preferred prokaryotic hosts include bacteria such asE. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, etc.Under such conditions, the Jagged will not be glycosylated. Theprokaryotic host must be compatible with the replicon and controlsequences in the expression plasmid.

However, prokaryotic systems may not prove efficacious for theexpression of a soluble Jagged ligand, since the protein of interestcontains 1045 residues encompassing residue 22 (after the signalsequence) to residue 1067 (prior to the transmembrane domain). Whileprokaryotic expression systems, e.g., pET3c, have been used to expresshigh molecular weight proteins, such as a biologically active (molecularweight (M_(r)) approximately 118 kDa) FGF-1:β-galactosidase chimera (Shiet al., 1997, J. Biol. Chem. 272:1142-1147), successful folding anddisulfide bond formation for the multiple EGF repeats (three disulfidebonds per EGF repeat) in the Jagged sequence may be difficult toaccomplish in bacteria.

Nevertheless, to express Jagged (or a functional derivative thereof) ina prokaryotic cell, it is necessary to operably link the Jagged codingsequence to a functional prokaryotic promoter. Such promoters may beeither constitutive or, more preferably, regulatable (i.e., inducible orderepressible). Examples of constitutive promoters include the intpromoter of bacteriophage λ, the bla promoter of the β-lactamase genesequence of pBR322, and the CAT promoter of the chloramphenicol acetyltransferase gene sequence of pPR325, etc. Examples of inducibleprokaryotic promoters include the major right and left promoters ofbacteriophage λ (P_(L) and P_(R)), the trp, reca, lacZ, lacI, and galpromoters of E. coli, the α-amylase (Ulmanen et al., 1985, J. Bacteriol.162:176-182) and the ζ-28-specific promoters of B. subtilis (Gilman etal., 1984, Gene Sequence 32:11-20), the promoters of the bacteriophagesof Bacillus (Gryczan, 1982, In: The Molecular Biology of the Bacilli,Academic Press, Inc., NY), and Streptomyces promoters (Ward et al.,1986, Mol. Gen. Genet. 203:468-478). See also reviews by Glick (1987, J.Ind. Microbiol. 1:277-282), Cenatiempo (1986, Biochimie 68:505-516), andGottesman (1984, Ann. Rev. Genet. 18:415-442).

Proper expression in a prokaryotic cell also requires the presence of aribosome binding site upstream of the gene sequence-encoding sequence.Such ribosome binding sites are disclosed, for example, by Gold et al.(1981, Ann. Rev. Microbiol. 35:365-404).

Preferred eukaryotic hosts include yeast, fungi, insect cells, mammaliancells, either in vivo or in tissue culture. Mammalian cells which may beuseful as hosts include HeLa cells, cells of fibroblast origin such asVERO or CHO-K1, or cells of lymphoid origin, such as the hybridomaSP2/O-AG14 or the myeloma P3x63Sg8, and their derivatives. Preferredmammalian host cells include SP2/0 and J558L, as well as neuroblastomacell lines such as IMR 332 that may provide better capacities forcorrect post-translational processing.

For a mammalian host, several possible vector systems are available forthe expression of Jagged. A wide variety of transcriptional andtranslational regulatory sequences may be employed, depending upon thenature of the host. The transcriptional and translational regulatorysignals may be derived from viral sources, such as adenovirus, bovinepapilloma virus, Simian virus, or the like, where the regulatory signalsare associated with a particular gene sequence which has a high level ofexpression. Alternatively, promoters from mammalian expression products,such as actin, collagen, myosin, etc., may be employed. Transcriptionalinitiation regulatory signals may be selected which allow for repressionor activation, so that expression of the gene sequences can bemodulated. Of interest are regulatory signals which aretemperature-sensitive so that by varying the temperature, expression canbe repressed or initiated, or are subject to chemical (such asmetabolite) regulation.

Yeast expression systems can also carry out post-translational peptidemodifications. A number of recombinant DNA strategies exist whichutilize strong promoter sequences and high copy number of plasmids whichcan be utilized for production of the desired proteins in yeast. Yeastrecognizes leader sequences on cloned mammalian gene sequence productsand secretes peptides bearing leader sequences (i.e., pre-peptides). Anyof a series of yeast gene sequence expression systems incorporatingpromoter and termination elements from the actively expressed genesequences coding for glycolytic enzymes produced in large quantitieswhen yeast are grown in mediums rich in glucose can be utilized. Knownglycolytic gene sequences can also provide very efficienttranscriptional control signals. For example, the promoter andterminator signals of the phosphoglycerate kinase gene sequence can beutilized.

The more preferred host for a protein the size of Jagged is insectcells, for example the Drosophila larvae. Using insect cells as hosts,the Drosophila alcohol dehydrogenase promoter can be used (see, e.g.,Rubin, 1988, Science 240:1453-1459).

The baculovirus insect cell expression system is the most preferredsystem for expressing the soluble Jagged construct (residues 1-1069) asa carboxy-terminal triple tandem myc-epitoperepeat:glutathione-S-transferase (GST) fusion protein chimera, usingconventional PCR methods (Zhan et al., 1994, J. Biol. Chem.269:20221-20224). These include the use of recombinant circle PCR tosynthesize the soluble Jagged-Myc-GST construct (sJMG), the preparationand expression of the recombinant virus, AcNPV-GsJ in Sf9 cells (Summersand Smith, 1988, In: A Manual of Methods for Baculovirus Vectors andInsect Culture Procedures, Texas Experimental Station Bulletin #1555),the use of GST affinity chromatography (Zhan et al., 1994) and reversedphase or ion exchange HPLC to purify the recombinant protein from Sf9cell lysates and Myc immunoblot analysis to monitor the purification andassess the purity of the sJMG protein.

As more fully set forth elsewhere herein, the sJMG construct may notonly prove to be valuable for the baculovirus expression system, but itis a useful construct for the expression of a secreted and solubleextracellular Jagged ligand in mammalian cells for implantation in vivo.Thus, the sJM construct—lacking the GST fusion domain—was inserted intothe pMEXneo vector and stable NIH 3T3 cell transfectants were obtainedfollowing selection with G418 as described (Zhan et al., 1992, Biochem.Biophys. Res. Commun. 188:982-991).

Indeed, in one embodiment, a nucleic acid encoding a soluble Jagged wasinserted into the pMEXneo vector and was used to successfully transfectNIH 3T3 cells. When injected into athymic nude mice, the transfectantsformed tissue masses demonstrating prominent angiogenesis. Further, thesoluble Jagged transfectants demonstrated altered growth morphology invitro forming chord-like structures when plated in plastic dishes in thepresence or absence of a collagen matrix. Further, soluble Jaggedtransfectants demonstrated a prominent angiogenic response onchorioallantoic membrane angiogenic (CAM) assays.

The soluble Jagged construct disclosed herein is missing both theintracellular and transmembrane domains of the full-length Jaggedprotein. However, the present invention should not be construed to belimited to constructs wherein both the transmembrane and intracellulardomains are not present in the protein molecule. Rather, the presentinvention encompasses constructs wherein a certain portion of the Jaggedprotein is absent whereby the truncated Jagged protein is bound to thecell membrane to a lesser extent than the full-length protein such thata greater amount of the truncated molecule in present in theextracellular milieu than the full-length protein.

Therefore, although the present invention discloses a truncated solubleJagged protein comprising from about amino acid residue 10 to aboutresidue 1180 of the full length protein, the invention is not limitedsolely to soluble Jagged containing these amino acid residues. Instead,Jagged protein comprising fewer or greater amino acid residues areencompassed in the soluble Jagged proteins of the invention.

Further, the present invention includes a soluble Jagged proteincomprising a tag epitope such as a myc tag epitope. By “tag epitope” ismeant any amino acid sequence, or nucleic acid encoding same, which,when linked by a peptide bond to a protein of interest, may be used tolocalize the protein, to purify it from a cell extract, to immobilize itfor use in binding assays, or to otherwise study its biologicalproperties and/or function.

One skilled in the art would appreciate, based upon the disclosureprovided herein, that the particular tag epitope which may form part ofthe soluble Jagged, is not limited to any particular tag epitope. Thatis, although the present invention includes covalently linking a nucleicacid encoding a myc epitope tag at the 3′ end of the nucleic acidencoding the truncated soluble Jagged protein, other tag epitopes suchas hemagglutinin, glutathione-S-transferase, myc-pyruvate kinase(myc-PK), His6, maltose biding protein (MBP), and the like, are includedin the invention. Thus, any nucleic acid sequence encoding a polypeptidewhich may function in a manner substantially similar to these tagpolypeptides should be included in the present invention.

Moreover, baculovirus vectors can be engineered to express large amountsof Jagged in insect cells (Jasny, 1987, Science 238:1653; Miller et al.,1986, In: Genetic Engineering, vol. 8, pp. 277-297, Setlow et al., eds.,Plenum Press).

As discussed above, expression of Jagged in eukaryotic hosts requiresthe use of eukaryotic regulatory regions. Such regions will, in general,include a promoter region sufficient to direct the initiation of RNAsynthesis. Preferred eukaryotic promoters include: the promoter of themouse metallothionein I gene sequence (Hamer et al., 1982, J. Mol. Appl.Gen. 1:273-288); the TK promoter of Herpes virus (McKnight, 1982, Cell31:355-365); the SV40 early promoter (Benoist et al., 1981, Nature290:304-310); the yeast gal4 gene sequence promoter (Johnston et al.,1982, Proc. Natl. Acad. Sci. USA 79:6971-6975; Silver et al., 1984,Proc. Natl. Acad. Sci. USA 81:5951-5955).

As is widely known, translation of eukaryotic mRNA is initiated at thecodon which encodes the first methionine. For this reason, it ispreferable to ensure that the linkage between a eukaryotic promoter anda DNA sequence which encodes Jagged (or a functional derivative thereof)does not contain any intervening codons which are capable of encoding amethionine (i.e., AUG). The presence of such codons results either in aformation of a fusion protein (if the AUG codon is in the same readingframe as the Jagged coding sequence) or a frame-shift mutation (if theAUG codon is not in the same reading frame as the Jagged codingsequence).

The Jagged coding sequence and an operably linked promoter may beintroduced into a recipient prokaryotic or eukaryotic cell either as anon-replicating DNA (or RNA) molecule, which may either be a linearmolecule or, more preferably, a closed covalent circular molecule. Sincesuch molecules are incapable of autonomous replication, the expressionof the Jagged may occur through the transient expression of theintroduced sequence. Alternatively, permanent expression may occurthrough the integration of the introduced sequence into the hostchromosome.

In one embodiment, a vector is employed which is capable of integratingthe desired gene sequences into the host cell chromosome. Cells whichhave stably integrated the introduced DNA into their chromosomes can beselected by also introducing one or more markers which allow forselection of host cells which contain the expression vector. The markermay provide for prototrophy to an auxotrophic host, biocide resistance,e.g., antibiotics, or heavy metals, such as copper, or the like. Theselectable marker gene sequence can either be directly linked to the DNAgene sequences to be expressed, or introduced into the same cell byco-transfection. Additional elements may also be needed for optimalsynthesis of single chain binding protein mRNA. These elements mayinclude splice signals, as well as transcription promoters, enhancers,and termination signals. cDNA expression vectors incorporating suchelements include those described by Okayama (1983, Molec. Cell. Biol.3:280-291).

In a preferred embodiment, the introduced sequence is incorporated intoa plasmid or viral vector capable of autonomous replication in therecipient host. Any of a wide variety of vectors may be employed forthis purpose. Factors of importance in selecting a particular plasmid orviral vector include: the ease with which recipient cells that containthe vector may be recognized and selected from those recipient cellswhich do not contain the vector; the number of copies of the vectorwhich are desired in a particular host; and whether it is desirable tobe able to “shuttle” the vector between host cells of different species.

Preferred prokaryotic vectors include plasmids, such as those capable ofreplication in E. coli (such as, for example, pBR322, ColE1, pSC101,pACYC 184, πVX. Such plasmids are, for example, disclosed by Maniatis etal. (1982, In: Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Press, Cold Spring Harbor, N.Y.). Bacillus plasmids includepC194, pC221, pT127, etc. Such plasmids are disclosed by Gryczan (1982,In: The Molecular Biology of the Bacilli, pp. 307-329, Academic Press,NY). Suitable Streptomyces plasmids include pIJ101 (Kendall et al.,1987, J. Bacteriol. 169:4177-4183), and streptomyces bacteriophages suchas φC31 (Chater et al., 1986, In: Sixth International Symposium onActinomycetales Biology, pp. 45-54, Akademiai Kaido, Budapest, Hungary).Pseudomonas plasmids are reviewed by John et al. (1986, Rev. Infect.Dis. 8:693-704), and Izaki (1978, Jpn. J. Bacteriol. 33:729-742).

Preferred eukaryotic plasmids include BPV, vaccinia, SV40, 2-microncircle, etc., or their derivatives. Such plasmids are well known in theart (Botstein et al., 1982, Miami Wntr. Symp. 19:265-274; Broach, 1981,In: The Molecular Biology of the Yeast Saccharomyces: Life Cycle andInheritance, pp. 445-470, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.; Broach, 1982, Cell 28:203-204; Bollon et al., 1980, J.Clin. Hematol. Oncol. 10:39-48; Maniatis, 1980, “Gene SequenceExpression,” In: Cell Biology: A Comprehensive Treatise, vol. 3, pp.563-608, Academic Press, NY).

Once the vector or DNA sequence containing the construct(s) has beenprepared for expression, the DNA construct(s) may be introduced into anappropriate host cell by any of a variety of suitable means:transformation, transfection, conjugation, protoplast fusion,electroporation, calcium phosphaie-precipitation, direct microinjection,etc. After the introduction of the vector, recipient cells are grown ina selective medium, which selects for the growth of vector-containingcells. Expression of the cloned gene sequence(s) results in theproduction of Jagged, or fragments thereof. This can take place in thetransformed cells as such, or following the induction of these cells todifferentiate (for example, by administration of bromodeoxyuracil toneuroblastoma cells or the like).

The Jagged proteins (or a functional derivatives thereof) of the presentinvention can be used in a variety of procedures and methods, such asfor the generation of antibodies, for use in identifying pharmaceuticalcompositions, and for studying DNA/protein interaction.

The peptides of the present invention may also be administered to amammal intravenously, intramuscularly, subcutaneously, enterally,topically or parenterally. When administering peptides by injection, theadministration may be by continuous injections, or by single or multipleinjections. The peptides are intended to be provided to a recipientmammal in a “pharmacologically or pharmaceutically acceptable form” inan amount sufficient to “therapeutically effective.” A peptide isconsidered to be in “pharmaceutically or pharmacologically acceptableform” if its administration can be tolerated by a recipient patient. Anamount is said to be “therapeutically effective” (an “effective amount”)if the dosage, route of administration, etc., of the agent aresufficient to affect a response to Jagged. Thus, the present peptidescan be used to increase or enhance the effect of the Jagged protein.

In another embodiment of the present invention, methods for inhibiting,decreasing or preventing the activity of the Jagged peptide can beachieved by providing an agent capable of binding to the ligand (or afunctional derivative thereof). Such agents include, but are not limitedto: antisense Jagged, the antibodies to Jagged (anti-Jagged), and thesecondary or anti-peptide peptides of the present invention. Bydecreasing the activity of Jagged, the affects which the expression ofthe peptide has on angiogenesis or restenosis can be modified.

In one example of the present invention, methods are presented fordecreasing the expression of Jagged (or a functional derivative thereof)by means of an anti-sense strand of cDNA to disrupt the translation ofthe Jagged message. Specifically, a cell is modified using routineprocedures such that it expresses an antisense message, a message whichis complementary to the pseudogene message. By constitutively orinducibly expressing the antisense RNA, the translation of the JaggedmRNA can be regulated. Such antisense technology has been successfullyapplied to regulate the expression of poly(ADP-ribose) polymerase (seeDing et al., 1992, J. Biol. Chem. 267:12804-12812).

On the other hand, methods for stimulating, increasing or enhancing theactivity of the Jagged peptide can be achieved by providing an agentcapable of enhancing the binding capability or capacity of the ligand(or a functional derivative thereof), or by inhibiting or preventing asignal which would diminish or stop the expression of Jagged in thesystem. Such agents include, but are not limited to, the anti-antisenseJagged peptides of the present invention. By enhancing the activity ofJagged, the effect which the expression of the peptide has onangiogenesis or restenosis can also be modified.

In yet another embodiment, Jagged (or a functional derivative or variantthereof) can be used to produce antibodies or hybridomas. One skilled inthe art will recognize that if an antibody is desired that will bind toJagged, such a ligand would be generated as described above and used asan immunogen. The resulting antibodies are then screened for the abilityto bind Jagged. Additionally, the antibody can be screened for itsinability to bind Notch.

The antibodies utilized in the above methods can be monoclonal orpolyclonal antibodies, as well fragments of these antibodies andhumanized forms. Humanized forms of the antibodies of the presentinvention may be generated using one of the procedures known in the artsuch as chimerization or CDR grafting.

In general, techniques for preparing monoclonal antibodies are wellknown in the art (Campbell, 1984, In: Monoclonal Antibody Technology:Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, Amsterdam, The Netherlands; St. Groth et al., 1980,J. Immunol. Methods 35:1-21). For example, in one embodiment an antibodycapable of binding Jagged is generated by immunizing an animal with asynthetic polypeptide whose sequence is obtained from a region of theJagged protein.

Any animal (mouse, rabbit, etc.) which is known to produce antibodiescan be utilized to produce antibodies with the desired specificity,although because of the large size of the Jagged molecule, the rabbit ismore preferred. Methods for immunization are well known in the art. Suchmethods include subcutaneous or interperitoneal injection of thepolypeptide. One skilled in the art will recognize that the amount ofpolypeptide used for immunization will vary based on the animal which isimmunized, the antigenicity of the polypeptide and the site ofinjection.

The polypeptide may be modified or administered in an adjuvant in orderto increase the peptide antigenicity. Methods of increasing theantigenicity of a polypeptide are well known in the art. Such proceduresinclude coupling the antigen with a heterologous protein (such asglobulin or β-galactosidase) or through the inclusion of an adjuvantduring immunization.

For monoclonal antibodies, spleen cells from the immunized animals areremoved, fused with myeloma cells, such as SP2/0-Ag14 myeloma cells, andallowed to become monoclonal antibody producing hybridoma cells. Ahybridoma is an immortalized cell line which is capable of secreting aspecific monoclonal antibody.

Any one of a number of methods well known in the art can be used toidentify the hybridoma cell which produces an antibody with the desiredcharacteristics. These include screening the hybridomas with an ELISAassay, western blot analysis, or radioimmunoassay (Lutz et al., 1988,Exp. Cell Res. 175:109-124).

Hybridomas secreting the desired antibodies are cloned and the class andsubclass are determined using procedures known in the art (Campbell,1984, In: Monoclonal Antibody Technology: Laboratory Techniques inBiochemistry and Molecular Biology, Elsevier Science Publishers,Amsterdam, The Netherlands).

For polyclonal antibodies, antibody containing antisera is isolated fromthe immunized animal and is screened for the presence of antibodies withthe desired specificity using one of the above-described procedures.

Conditions for incubating an antibody with a test sample vary.Incubating conditions depend on the format employed in the assay, thedetection methods employed, the nature of the test sample, and the typeand nature of the antibody used in the assay. One skilled in the artwill recognize that any one of the commonly available immunologicalassay formats (such as, radioimmunoassays, enzyme-linked immunosorbentassays, diffusion based Ouchterlony, or rocket immunofluorescent assays,or the like) can readily be adapted to employ the antibodies of thepresent invention. Examples of such assays can be found in Chard (1986,In: An Introduction to Radioimmunoassay and Related Techniques, ElsevierScience Publishers, Amsterdam, The Netherlands; Bullock et al., In:Techniques in Immunocytochemistry, Academic Press, Orlando, Fla., vol. 1(1982), vol. 2 (1983), vol. 3 (1985); Tijssen, 1985, In: Practice andTheory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry andMolecular Biology, Elsevier Science Publishers, Amsterdam, TheNetherlands).

The anti-Jagged antibody is also effective when immobilized on a solidsupport. Examples of such solid supports include, but are not limitedto, plastics such as polycarbonate, complex carbohydrates such asagarose and sepharose, and acrylic resins, such as polyacrylamide andlatex beads. Techniques for coupling antibodies to such solid supportsare well known in the art (Weir et al., 1986, In: Handbook ofExperimental Immunology, chapter 10, 4th ed., Blackwell ScientificPublications, Oxford, England; Jacoby et al., 1974, In: Methods inEnzymology, vol. 34 Academic Press, N.Y.).

Additionally, one or more of the antibodies used in the above describedmethods can be detectably labeled prior to use. Antibodies can bedetectably labeled through the use of radioisotopes, affinity labels(such as, biotin, avidin, etc.), enzymatic labels (such as, horse radishperoxidase, alkaline phosphatase, etc.) fluorescent labels (such as,FITC or rhodamine, etc.), paramagnetic atoms, etc. Procedures foraccomplishing such labelling are well-known in the art (see, e.g.,Stemberger et al., 1970, J. Histochem. Cytochem. 18:315-333; Bayer etal., 1979, Meth. Enzym. 62:308-315; Engval et al., 1972, Immunol.109:129-; Goding, 1976, J. Immunol. Meth. 13:215-226). The labeledantibodies of the present invention can be used for, among other things,in vitro, in vivo, and in situ assays to identify cells or tissues whichexpress a specific protein or ligand.

In an embodiment of the above methods, the antibodies are labeled, suchthat a signal is produced when the antibody(s) bind to the samemolecule. One such system is described in U.S. Pat. No. 4,663,278.

The antibodies or antisense peptides of the present invention may beadministered to a mammal intravenously, intramuscularly, subcutaneously,enterally, topically or parenterally. When administering antibodies orpeptides by injection, the administration may be by continuousinjections, or by single or multiple injections.

The antibodies or antisense peptides of the present invention areintended to be provided to a recipient mammal in a “pharmaceuticallyacceptable form” in an amount sufficient to be “therapeuticallyeffective” or an “effective amount”. As above, an amount is said to betherapeutically effective (an effective amount), if the dosage, route ofadministration, etc. of the agent are sufficient to affect the responseto Jagged. Thus, the present antibodies may either stimulate or enhancethe effect of the Jagged protein, or they may inhibit or prevent theeffect of the Jagged protein. Or, secondary antibody(s) may be designedto affect the response to the Jagged antibody(s) per se, i.e., ananti-antibody to Jagged. In the alternative, either an antibody or ananti-antibody may be designed to affect only the anti-sense strand ofthe ligand.

One skilled in the art can readily adapt currently available proceduresto generate secondary antibody peptides capable of binding to a specificpeptide sequence in order to generate rationally designed antipeptidepeptides, for example see Hurby et al., 1992, “Application of SyntheticPeptides: Antisense Peptides”, In: Synthetic Peptides, A User's Guide,pp. 289-307, W.H. Freeman, NY; Kaspczak et al., 1989, Biochemistry28:9230-9238). As used herein, an agent is said to be “rationallyselected or designed” when the agent is chosen based on theconfiguration of the Jagged peptide.

Anti-peptide peptides can be generated in one of two fashions. First,the anti-peptide peptides can be generated by replacing the basic aminoacid residues found in the pseudogene peptide sequence with acidicresidues, while maintaining hydrophobic and uncharged polar groups. Forexample, lysine, arginine, and/or histidine residues are replaced withaspartic acid or glutamic acid and glutamic acid residues are replacedby lysine, arginine or histidine. Alternatively, the anti-peptidepeptides of the present invention can be generated by synthesizing andexpressing a peptide encoded by the antisense strand of the DNA whichencodes the pseudogene peptide. Peptides produced in this fashion are,in general, similar to those described above since codons complementaryto those coding for basic residues generally code for acidic residues.

To detect secondary antibodies, or in the alternative, the labeledprimary antibody, labelling reagents may include, e.g., chromophobic,enzymatic, or antibody binding reagents which are capable of reactingwith the labelled antibody. One skilled in the art will readilyrecognize that the disclosed antibodies of the present invention canreadily be incorporated into one of the established kit formats whichare well known in the art.

An antibody is said to be in “pharmaceutically or pharmacologicallyacceptable form” if its administration can be tolerated by a recipientpatient. The antibodies of the present invention can be formulatedaccording to known methods of preparing pharmaceutically usefulcompositions, whereby these materials, or their functional derivatives,are combined with a pharmaceutically acceptable carrier vehicle.Suitable vehicles and their formulation, inclusive of other humanproteins, e.g., human serum albumin, are described, for example, inRemington's Pharmaceutical Sciences, 1980.

In order to form a pharmaceutically acceptable composition which issuitable for effective administration, such compositions will contain aneffective amount of an antibody of the present invention together with asuitable amount of carrier. Such carriers include, but are not limitedto saline, buffered saline, dextrose, water, glycerol, ethanol, and acombination thereof. The carrier composition may be sterile. Theformulation should suit the mode of administration. In addition tocarriers, the antibodies of the present invention may be supplied inhumanized form.

Humanized antibodies may be produced, for example by replacing animmunogenic portion of an antibody with a corresponding, butnon-immunogenic portion (i.e., chimeric antibodies) (Robinson et al.,International Patent Publication PCT/US86/02269; Akira et al., EuropeanPatent Application 184,187; Taniguchi, European Patent Application171,496; Morrison et al., European Patent Application 173,494; Neubergeret al., PCT Application WO 86/01533; Cabilly et al., European PatentApplication 125,023; Better et al., 1988, Science 240:1041-1043; Liu etal., 1987, Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987, J.Immunol. 139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al.,1985, Nature 314:446-449; Shaw et al., 1988, J. Natl. Cancer Inst.80:1553-1559).

The compositions of the present invention can also include minor amountsof wetting or emulsifying agents, or pH buffering agents. Thecomposition can be a liquid solution, suspension, emulsion, tablet,pill, capsule, sustained release formulation or powder. The compositioncan be formulated as a suppository with traditional binders and carrierssuch as triglycerides. Oral formulations can include standard carrierssuch as pharmaceutically acceptable mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate, etc.

In a preferred embodiment of the present invention, the compositions areformulated in accordance with routine procedures for intravenousadministration to a subject. Typically, such compositions are carried ina sterile isotonic aqueous buffer. As needed, a composition may includea solubilizing agent and a local anesthetic. Generally, the componentsare supplied separately or as a mixture in unit dosage form, such as adry lyophilized powder in a sealed container with an indication ofactive agent. Where the composition is administered by infusion, it maybe provided with an infusion container with a sterile pharmaceuticallyacceptable carrier. When the composition is administered by injection,an ampoule of sterile water or buffer may be included to be mixed priorto injection.

The therapeutic compositions may also be formulated in salt form.Pharmaceutically acceptable salts include those formed with free aminogroups, such as those derived from hydrochloric, phosphoric, acetic,oxalic and tartaric acids, or formed with free carboxyl groups such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The dosage of the administered agent will vary depending upon suchfactors as the patient's age, weight, height, sex, general medicalcondition, previous medical history, etc. In general, it is desirable toprovide the recipient with a dosage of the antibody which is in therange of from about 1 pg/kg to 10 mg/kg (body weight of patient),although a lower or higher dosage may be administered. Suitable rangesfor intravenous administration is typically about 20-500 μg of activecompound per kilogram body weight. Effective doses may be extrapolatedfrom dose-response curves derived from in vitro and in vivo animal modeltest systems.

Since highly purified proteins are now available, X-ray crystallographyand NMR-imaging techniques can be used to identify the structure of theligand binding site. Utilizing such information, computer modelingsystems are now available that allow one to “rationally design” an agentcapable of binding to a defined structure (Hodgson, 1990, Biotechnology8:1245-1247; Hodgson, 1991, Biotechnology 9:609-613). As used herein, anagent is said to be “rationally designed” if it is selected based on acomputer model of the ligand or Notch binding site, or in thealternative, of the ligand binding site on Jagged if activation of theNotch binding site is found to act as an on/off switch affecting thecontinued expression of Jagged.

In another embodiment of the present invention, methods are provided formodulating the translation of RNA encoding Jagged protein in the cell.Specifically, said method comprises introducing into a cell a DNAsequence which is capable of transcribing RNA which is complimentary tothe RNA encoding the Jagged protein. By introducing such a DNA sequenceinto a cell, antisense RNA will be produced which will hybridize andblock the translation of the Jagged protein. Antisense cloning has beendescribed by Rosenberg et al. (1985, Nature 313:703-706), Preiss et al.(1985, Nature 313:27-32), Melton (1985, Proc. Natl. Acad. Sci. USA82:144-148), and Kim et al. (1985, Cell 42:129-138).

Transcription of the introduced DNA will result in multiple copies ofantisense RNA which will be complementary to the Jagged. By controllingthe level of transcription of antisense RNA, and the tissue specificityof expression, one skilled in the art can regulate the level oftranslation of Jagged protein in specific cells within a patient.

In one aspect of the above-described invention, DNA response elements(RE) can be identified which are capable of either stimulating orinhibiting the binding of Jagged. In this manner, assays may beperformed to determine binding agents by using any length of DNA so longas it contains at least one RE sequence. In another embodiment, theabove such assays are performed in the absence of a RE. In this fashion,agents can be identified which bind to or affect the binding capacity ofJagged independently of DNA binding. Moreover, the above assay can bemodified so that it is capable of identifying agents which activatetranscription of DNA sequences controlled by a RE.

In the present invention, a cell or organism is altered using routinemethods such that it expresses Jagged, or a functional derivativethereof. Moreover, the cell or organism may be further altered tocontain a RE operably linked to a reporter sequence, such as luciferase,beta galactosidase, or chloramphenicol acyltransferase. Agents are thenincubated with the cell or organism and the expression of the reportersequence is assayed.

In an alternative usage, nuclear and/or cytosolic extracts from thealtered cell containing Jagged or a functional derivative thereof aremixed with an expression module containing an RE operably linked to areporter sequence. The extract/expression module is incubated with anagent and the expression of the reporter sequence is assayed.

Isolated Nucleic Acid Encoding Soluble Jagged

The invention includes an isolated nucleic acid encoding a solubleJagged protein. Preferably, the nucleic acid encoding a soluble Jaggedis at least about 20% homologous to a nucleic acid having the nucleicacid sequence of SEQ ID NO:17 which is depicted in FIGS. 13B and 13C.The nucleic acid encoding soluble Jagged (SEQ ID NO:17) comprises fromabout nucleotide 1 to about nucleotide 3201 of full-length Jaggedsequence (GenBank Acc. No. U77720, [SEQ ID NO:2]), which sequence isdepicted in FIGS. 8B and 8C.

More preferably, the isolated nucleic acid encoding a soluble Jagged isat least about 20% homologous, more preferably, at least about 30%,homologous, preferably, at least about 40%, more preferably, at leastabout 50%, even more preferably, at least about 60%, more preferably, atleast about 70%, even more preferably, at least about 80%, yet morepreferably, at least about 90% homologous, more preferably, at leastabout 95% and even more preferably, at least about 99% homologous to(SEQ ID NO:17). More preferably, the isolated nucleic acid encoding asoluble Jagged is soluble Jagged-1. Most preferably, the isolatednucleic acid encoding a soluble Jagged is SEQ ID NO:17.

The invention also includes a nucleic acid encoding a soluble Jagged, ora fragment or portion thereof. That is, the invention encompasses anucleic acid encoding less than the full-length soluble Jagged disclosedherein. This is because one skilled in the art would appreciate, basedupon the disclosure provided herein, that a nucleic acid encoding lessthan the full-length soluble Jagged can be useful for a variety ofpurposes included providing portions of the protein for use in antibodyproduction, treatments related to inhibiting Jagged/Notch interactions,repressing expression of type I collagen (which is extremely importantin the regulation of fibrotic diseases), and the like.

As used herein, the term “fragment” as applied to a nucleic acidencoding a soluble Jagged, may ordinarily be at least about 30nucleotides in length, typically, at least about 50 nucleotides, moretypically, from about 50 to about 100 nucleotides, preferably, at leastabout 100 to about 500 nucleotides, even more preferably, at least about500 nucleotides to about 1000 nucleotides, yet even more preferably atleast about 1000 to about 1500, more preferably, at least about 1500 toabout 2500 nucleotides, even more preferably, at least about 2500nucleotides to about 3000 nucleotides, yet even more preferably, atleast about 3000 to about 3100, more preferably, at least about 3100 toabout 3160, yet more preferably, at least about 3160 to about 3200, andmost preferably, the nucleic acid fragment will be greater than about3200 nucleotides in length.

As applied to a protein, a soluble Jagged “fragment” is about 30 aminoacids in length. More preferably, the fragment is about 40 amino acids,even more preferably, at least about 100, yet more preferably, at leastabout 200, even more preferably, at least about 500, yet morepreferably, at least about 750, even more preferably, at least about800, yet more preferably, at least about 850, more preferably, at leastabout 900, yet more preferably, at least about 950, even morepreferably, at least about 1000, yet more preferably, at least about1050, yet more preferably, at least about 1060, and more preferably, atleast about 1060 amino acids in length.

The invention includes a nucleic acid encoding a soluble Jagged whereinoptimally a nucleic acid encoding a tag polypeptide is covalently linkedthereto. That is, the invention encompasses a chimeric nucleic acidwherein a nucleic acid sequence encoding a tag polypeptide is covalentlylinked to a nucleic acid encoding soluble Jagged. Such chimeric (i.e.,fusion) tag polypeptides are well known in the art and include, forinstance, myc, myc-pyruvate kinase (myc-PK), His₆, maltose bidingprotein (MBP), glutathione-S-transferase (GST), and green fluorescenceprotein (GFP). However, the invention is not limited to the nucleicacids encoding the above-listed tag polypeptides. Rather, any nucleicacid sequence encoding a polypeptide which can function in a mannersubstantially similar to these tag polypeptides should be construed tobe included in the present invention. Further, more than one tagpolypeptide can be expressed along with a nucleic acid encoding aprotein of interest. That is, one skilled in the art would understand,based upon the disclosure provided herein, that more than one tagpolypeptide can be covalently linked with a soluble Jagged protein.

A nucleic acid encoding a protein of interest (e.g., soluble Jagged, andany mutant, derivative, variant, or fragment thereof) comprising anucleic acid encoding a tag polypeptide and a fusion protein producedtherefrom can be used to, among other things, localize soluble Jaggedwithin a cell and to study expression, localization, and role(s) of thetagged protein in a cell before, during, and/or after exposing the cellto a test compound. Further, addition of a tag to a protein of interestfacilitates isolation and purification of the “tagged” protein such thatthe protein of interest can be easily produced and purified.

In other related aspects, the invention includes a nucleic acid encodinga soluble Jagged operably linked to a nucleic acid comprising apromoter/regulatory sequence such that the nucleic acid is preferablycapable of directing expression of the protein encoded by the nucleicacid.

Expression of soluble Jagged, either alone or fused to a detectable tagpolypeptide, in cells which either do not normally express solubleJagged or which do not express soluble Jagged comprising a tagpolypeptide, can be accomplished by operably linking the nucleic acidencoding soluble Jagged to a promoter/regulatory sequence which servesto drive expression of the protein, with or without a tag polypeptide,in a cell into which the exogenous nucleic acid is introduced.

As disclosed previously elsewhere herein, many promoter/regulatorysequences useful for driving constitutive expression of a gene areavailable in the art and include, but are not limited to, for example,the cytomegalovirus immediate early promoter enhancer sequence, the SV40early promoter, both of which were used in the experiments disclosedherein, as well as the Rous sarcoma virus promoter, and the like.Moreover, inducible and tissue specific expression of the nucleic acidencoding soluble Jagged can be accomplished by placing the nucleic acidencoding soluble Jagged, with or without a tag polypeptide, under thecontrol of an inducible or tissue specific promoter/regulatory sequence.Examples of tissue specific or inducible promoter/regulatory sequenceswhich are useful for his purpose include, but are not limited to theMMTV LTR inducible promoter, and the SV40 late enhancer/promoter. Inaddition, promoters which are well known in the art which are induced inresponse to inducing agents such as metals, glucocorticoids, and thelike, are also contemplated in the invention. Thus, it will beappreciated that the invention includes the use of anypromoter/regulatory sequence, which is either known or unknown, andwhich is capable of driving expression of the desired protein encoded bya nucleic acid operably linked to the promoter/regulatory sequence.

Expressing soluble Jagged using a promoter/regulatory sequence allowsthe isolation of large amounts of recombinantly produced protein.Further, where the lack or decreased level of soluble Jagged expressioncauses a disease, disorder, or condition associated with suchexpression, the expression of the protein driven by apromoter/regulatory sequence can provide useful therapeutics including,but not limited to, gene therapy whereby the protein is provided.

Vectors

The invention also includes a vector comprising a nucleic acid encodinga soluble Jagged. Methods for incorporating a desired nucleic acid intoa vector and the choice of vectors is well-known in the art as describedin, for example, Sambrook et al.,.supra, and Ausubel et al., supra, andare disclosed elsewhere herein.

Further, the invention encompasses expression vectors and methods forthe introduction of exogenous nucleic acid encoding soluble Jagged intoa cell with concomitant expression of the exogenous nucleic acid in thecell using such methods as those described in, for example, Sambrook etal. (1989, supra), and Ausubel et al. (1997, supra), and as disclosedelsewhere herein.

Selection of any particular plasmid vector or other DNA vector is not alimiting factor in this invention and a wide plethora vectors arewell-known in the art (see, e.g., Sambrook et al., supra, and Ausubel etal., supra.). Further, it is well within the skill of the artisan tochoose particular promoter/regulatory sequences and operably link thosepromoter/regulatory sequences to a DNA sequence encoding a desiredpolypeptide. Such technology is well known in the art and is described,for example, in Sambrook, supra, and Ausubel, supra.

The invention also includes cells, viruses, proviruses, and the like,containing such vectors. Methods for producing cells comprising vectorsand/or exogenous nucleic acids are well-known in the art. See, e.g.,Sambrook et al., supra; Ausubel et al., supra.

The nucleic acids encoding soluble Jagged can be cloned into variousplasmid vectors. However, the present invention should not be construedto be limited to plasmids or to any particular vector. Instead, thepresent invention should be construed to encompass a wide plethora ofvectors which are readily available and/or well-known in the art.

Recombinant Cells

Additionally, the nucleic and amino acids of the invention can be usedto produce recombinant cells which are useful tools for the study ofsoluble Jagged, the identification of novel soluble Jagged-basedtherapeutics, and for elucidating the cellular role(s) of solubleJagged, among other things.

Further, the nucleic and amino acids of the invention can be useddiagnostically, by assessing either the level of gene expression orprotein expression and the biological activity of the protein, to assessseverity and prognosis of a disease, disorder, or condition associatedwith altered level of soluble Jagged gene expression.

The invention also includes expression of soluble Jagged in a cell whereit is not normally expressed or expression of soluble Jagged-taggedfusion protein in cells where this fusion protein is not normallyexpressed. In a preferred embodiment, nucleic acid encoding solubleJagged was covalently linked with a nucleic acid expressing a tagpolypeptide and used to transfect a mammalian cell. Plasmid constructscontaining soluble Jagged, or mutants, variants, derivatives andfragments thereof, can be cloned into a wide variety of vectorsincluding a vector comprising a nucleic acid encoding a tag polypeptide.The plasmids can be introduced into a cell using standard methodswell-known in the art (e.g., calcium phosphate, electroporation, and thelike). Methods for cloning and introducing an isolated nucleic acid ofinterest into a cell are exemplified herein and are described in, forexample, Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, New York), Ausubel et al. (1997,Current Protocols in Molecular Biology, Green & Wiley, New York), andother standard treatises.

The present invention also encompasses expression of an isolated solubleJagged of the invention in non-mammalian cells (e.g. yeast, insect, andavian cells) using methods well-known in the art such as those disclosedelsewhere herein. Thus, it is clear that the invention is not limited toany particular vector or to any particular method of introducing theexogenous nucleic acid encoding soluble Jagged into a cell.

Expression of proteins of interest (e.g., soluble Jagged) in a cell,especially when the protein comprises a tag polypeptide, allowslocalization of the nucleic acid and/or the protein expressed therefromwithin the cell under selected conditions such that the function(s) ofthe protein in the cell can be studied and identified.

One skilled in the art would appreciate, based upon the disclosureprovided herein, that the invention also includes expression of solubleJagged, and the like, in prokaryotic cells (e.g., bacterial cells suchas, for example, E. coli ). Accordingly, the invention includesexpression of the proteins of the invention in such cells as well.

The invention should not be construed as being limited to these plasmidvectors, bacterial strains, or to these tag polypeptides. Further, theinvention is not limited to calcium phosphate transfection or to NIHcells as exemplified herein. Instead, the invention encompasses otherexpression vectors and methods for the introduction of exogenous DNAinto cells with concomitant expression of the exogenous DNA in the cellssuch as those described, for example, in Sambrook et al. (1989, supra),and Ausubel et al. (1997, supra).

In one embodiment, the cell line is mammalian cell comprising anexpression vector comprising a nucleic acid encoding soluble Jaggedconstitutively expressed under the control of a high-level expressionpromoter/regulatory sequence. Further, the skilled artisan wouldappreciate based upon the disclosure provided herein that the cells canbe transfected with constructs which comprise soluble Jagged in either asense (i.e., sense cells) or an antisense orientation (i.e., antisensecells).

One skilled in the art would further appreciate that selected forms ofnucleic acids encoding soluble Jagged can be introduced to a cell inorder to study the effect of any mutant, derivative, and variant ofsoluble Jagged (e.g., fusion proteins comprising at least a portion ofsoluble Jagged and a tag polypeptide) in this system.

Further, the invention includes a recombinant cell comprising anantisense nucleic acid (e.g., γ-soluble Jagged) which cell is a usefulmodel for the study of a disease, disorder, or condition associated withor mediated by inhibition of soluble Jagged biosynthesis and forelucidating the role(s) of soluble Jagged in such processes. That is,the lack of expression of soluble Jagged in patients may indicate, amongother things, a disease, disorder or condition. Accordingly, arecombinant (i.e., transgenic) cell comprising an antisense nucleic acidcomplementary to a nucleic acid encoding soluble Jagged is a useful toolfor the study of the mechanism(s) of action of soluble Jagged and itsrole(s) in the cell and for the identification of therapeutics thatameliorate the effect(s) of decreased levels of soluble Jaggedexpression.

The invention further includes a recombinant cell comprising an isolatednucleic acid encoding soluble Jagged. The cell can be transientlytransfected with a plasmid encoding a portion of the nucleic acidencoding the protein of interest, e.g., soluble Jagged. The nucleic acidneed not be integrated into the cell genome nor does it need to beexpressed in the cell. Moreover, the cell may be a prokaryotic or aeukaryotic cell and the invention should not be construed to be limitedto any particular cell line or cell type.

When the cell is a eukaryotic cell, the cell may be any eukaryotic cellwhich, when the isolated nucleic acid of the invention is introducedtherein, and the protein encoded by the desired gene is no longerexpressed therefrom, a benefit is obtained. Such a benefit may includethe fact that there has been provided a system in which lack ofexpression of the desired gene can be studied in vitro in the laboratoryor in a mammal in which the cell resides, a system wherein cellscomprising the introduced gene deletion can be used as research,diagnostic and therapeutic tools, and a system wherein animal models aregenerated which are useful for the development of new diagnostic andtherapeutic tools for selected disease, disorder, or condition states ina mammal.

Alternatively, the invention includes a eukaryotic cell which, when theisolated nucleic acid of the invention is introduced therein, and theprotein encoded by the desired gene, i.e., soluble Jagged, is expressedtherefrom where it was not previously present or expressed in the cellor where it is now expressed at a level or under circumstances differentthan that before the isolated nucleic acid was introduced, a benefit isobtained. Such a benefit may include the fact that there has beenprovided a system wherein the expression of the desired gene can bestudied in vitro in the laboratory or in a mammal in which the cellresides, a system wherein cells comprising the introduced gene can beused as research, diagnostic and therapeutic tools, and a system whereinanimal models are generated which are useful for the development of newdiagnostic and therapeutic tools for selected disease states in a mammal(e.g., diseases, disorders or conditions of the pituitary mediated byaltered expression or activity of soluble Jagged).

Isolated Polypeptides

The invention includes an isolated polypeptide encoded by a nucleic acidencoding a soluble Jagged where the amino acid sequence of thepolypeptide is preferably, at least about 30% homologous to the aminoacid sequence of soluble Jagged (SEQ ID NO:18). More preferably, theisolated nucleic acid encodes a soluble Jagged which is at least about40%, more preferably, at least about 50%, even more preferably, at leastabout 60%, yet more preferably, at least about 70%, more preferably, atleast about 80%, even more preferably, at least about 90%, yet morepreferably, at least about 95%, and even more preferably, at least about99% homologous to (SEQ ID NO:18). More preferably, the isolated nucleicacid encodes a soluble Jagged that is soluble Jagged. Most preferably,the isolated nucleic acid encodes a soluble Jagged having the amino acidsequence SEQ ID NO:18.

The invention also includes an isolated polypeptide comprising a solubleJagged. Preferably, the isolated polypeptide comprising a mammaliansoluble Jagged is at least about 30% homologous to SEQ ID NO:18. Morepreferably, the isolated polypeptide comprising a mammalian solubleJagged is at least about 40%, more preferably, at least about 50%, evenmore preferably, at least about 60%, yet more preferably, at least about70%, more preferably, at least about 80%, even more preferably, at leastabout 90%, yet more preferably, at least about 95%, and more preferably,at least about 99% homologous to soluble Jagged (SEQ ID NO:18). Morepreferably, the isolated polypeptide is soluble Jagged. Most preferably,the isolated polypeptide comprising a soluble Jagged is SEQ ID NO:18.

The invention also includes an isolated polypeptide comprising a portionof Jagged. Preferably, the isolated polypeptide comprising a portion ofJagged is at least about 30% homologous to SEQ ID NO:18. Morepreferably, the isolated polypeptide comprising a portion of solubleJagged is at least about 40%, more preferably, at least about 50%, evenmore preferably, at least about 60%, yet more preferably, at least about70%, more preferably, at least about 80%, even more preferably, at leastabout 90%, yet more preferably, at least about 95%, and more preferably,at least about 99% homologous to SEQ ID NO:18. More preferably, theisolated polypeptide comprising a portion of soluble Jagged is a portionof Jagged (SEQ ID NO:1) (e.g., from about amino acid 1 to about aminoacid 1067). Most preferably, the isolated polypeptide comprising aportion of Jagged is SEQ ID NO:18.

The present invention also provides for analogs of proteins or peptideswhich comprise a soluble Jagged protein as disclosed herein. Analogs maydiffer from naturally occurring proteins or peptides by conservativeamino acid sequence differences or by modifications which do not affectsequence, or by both. For example, conservative amino acid changes maybe made, which although they alter the primary sequence of the proteinor peptide, do not normally alter its function. Conservative amino acidsubstitutions typically include substitutions within the followinggroups:

glycine, alanine;

valine, isoleucine, leucine;

aspartic acid, glutamic acid;

asparagine, glutamine;

serine, threonine;

lysine, arginine;

phenylalanine, tyrosine;

Modifications (which do not normally alter primary sequence) include invivo, or in vitro, chemical derivatization of polypeptides, e.g.,acetylation, or carboxylation. Also included are modifications ofglycosylation, e.g., those made by modifying the glycosylation patternsof a polypeptide during its synthesis and processing or in furtherprocessing steps e.g., by exposing the polypeptide to enzymes whichaffect glycosylation, e.g., mammalian glycosylating or deglycosylatingenzymes. Also embraced are sequences which have phosphorylated aminoacid residues, e.g., phosphotyrosine, phosphoserine, orphosphothreonine.

Also included are polypeptides which have been modified using ordinarymolecular biological techniques so as to improve their resistance toproteolytic degradation or to optimize solubility properties or torender them more suitable as a therapeutic agent. Analogs of suchpolypeptides include those containing residues other than naturallyoccurring L-amino acids, e.g., D-amino acids or non-naturally occurringsynthetic amino acids. The peptides of the invention are not limited toproducts of any of the specific exemplary processes listed herein.

The present invention should also be construed to encompass “mutants,”“derivatives,” and “variants” of the peptides of the invention (or ofthe DNA encoding the same) which mutants, derivatives and variants aresoluble Jagged polypeptides which are altered in one or more amino acids(or, when referring to the nucleotide sequence encoding the same, arealtered in one or more base pairs) such that the resulting peptide (orDNA) is not identical to the sequences recited herein, but has the samebiological property as the peptides disclosed herein, in that thepeptide has biological/biochemical properties of the soluble Jaggedprotein of the present invention. A biological property of a solubleJagged includes, but is not limited to include, the ability of thepeptide to bind specifically with Notch as demonstrated using, forexample, electrophoretic mobility shift assay (EMSA) as disclosedelsewhere herein. Further, another biological activity of soluble Jaggedis the ability to affect the level of expression of various nucleicacids enhancing expression of certain genes (e.g., enhancer of splitgroucho, type IV collagenase, connexin 32, cathepsin D, and vimentin),while mediating reduced levels of expression of other genes (e.g.,pro-α-2(I) collagen, FGFR-1, and IkB-β), as determined using serialanalysis of gene expression (SAGE) analysis. Further, the activities ofsoluble Jagged include, but are not limited to, affecting endothelialsprout formation, affecting angiogenesis, the ability to inducedevelopment of angiogenic tissue masses in nude mice, and affecting theability to induce angiogenesis in a CAM angiogenesis model.Additionally, the biological activity of soluble Jagged includes theability to repress type I collagen expression, which is extremelyimportant in the regulation of all fibrotic diseases.

Further, the invention should be construed to include naturallyoccurring variants or recombinantly derived mutants of soluble Jagged,which variants or mutants render the protein encoded thereby eithermore, less, or just as biologically active as the full-length proteinsand/or the truncated soluble proteins of the invention.

In addition, the skilled artisan would appreciate that changes can beintroduced by mutation of the nucleic acid encoding the protein therebyleading to changes in the amino acid sequence of the encoded protein,without altering the biological activity of the protein. For example,one can make nucleotide substitutions leading to amino acidsubstitutions at “non-essential” amino acid residues. A “non-essential”amino acid residue is a residue that can be altered from the wild-typesequence without altering the biological activity, whereas an“essential” amino acid residue is required for biological activity. Forexample, amino acid residues that are not conserved or onlysemi-conserved among homologs of various species may be non-essentialfor activity and thus would be likely targets for alteration.Alternatively, amino acid residues that are conserved among the homologsof various species (e.g., murine and human) may be essential foractivity and thus would not be likely targets for alteration.

Accordingly, another aspect of the invention pertains to polypeptidesencoded by nucleic acid molecules of the invention, which polypeptidescontain changes in amino acid residues that are not essential foractivity. Such polypeptides differ in amino acid sequence from any ofSEQ ID NOS:1, and SEQ ID NO:18, yet retain biological activity.

To generate variant proteins, an isolated nucleic acid molecule encodinga variant protein can be created by introducing one or more nucleotidesubstitutions, additions or deletions into the nucleotide sequence ofany of SEQ ID NO:2 and/or SEQ ID NO:17, such that one or more amino acidresidue substitutions, additions or deletions are introduced into theencoded soluble Jagged protein. Mutations can be introduced by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions are madeat one or more predicted non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),non-polar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively,mutations can be introduced randomly along all or part of the codingsequence, such as by saturation mutagenesis, and the resultant mutantscan be screened for biological activity to identify mutants that retainactivity. Following mutagenesis, the encoded protein can be expressedrecombinantly and the activity of the protein can be determined.

One skilled in the art would appreciate, based upon the disclosureprovided herein, that a mutant polypeptide that is a variant of apolypeptide of the invention can be assayed for: (1) the ability toinduce expression of certain genes (e.g., enhancer of split groucho,type IV collagenase, connexin 32, cathepsin D, and vimentin); (2) theability to reduce expression of various genes (e.g., pro-α-2(I)collagen, FGFR-1, and IkB-β); (3) the ability to induce sproutformation; (4) the ability to induce angiogenesis in a CMA model; (5)the ability to induce formation of angiogenic tissue masses in nudemice; and (6) the ability to repress type I collagen expression, whichis extremely important in the regulation of all fibrotic diseases.

The nucleic acids, and peptides encoded thereby, are useful tools forelucidating the function(s) soluble Jagged in a cell. Further, they areuseful for localizing the nucleic acid, protein, or both, in a cell andfor assessing the level of expression of the nucleic acid and/or proteinunder selected conditions including in response to therapeutictreatment. Further, nucleic and amino acids comprising soluble Jaggedare useful diagnostics which can be used, for example, to identify acompound that affects expression of the protein and is a candidatetherapeutic for a disease, disorder, or condition associated withaltered expression of soluble Jagged.

In addition, the nucleic acids, the proteins encoded thereby, or both,can be administered to a mammal to increase or decrease expression ofsoluble Jagged in the mammal. This can be therapeutic to the mammal ifunder or over-expression of soluble Jagged in the mammal mediates adisease or condition associated with altered expression of the proteincompared with normal expression of soluble Jagged in a healthy mammal.

Antibodies

The invention also includes an antibody specific for a soluble Jagged,or a portion thereof.

In one embodiment, the antibody is a rabbit polyclonal antibody tosoluble Jagged. The antibody can be specific for any portion of theprotein and the full-length protein can be used to generate antibodiesspecific therefor. However, the present invention is not limited tousing the full-length protein as an immunogen. Rather, the presentinvention includes using an immunogenic portion of the protein toproduce an antibody that specifically binds with soluble Jagged. Thatis, the invention includes immunizing an animal using an immunogenicportion of the protein.

The antibodies can be produced by immunizing an animal such as, but notlimited to, a rabbit or a mouse with a protein of the invention, or aportion thereof, or by immunizing an animal using a protein comprisingat least a portion of soluble Jagged and a tag polypeptide portioncomprising, for example; a maltose binding protein tag polypeptideportion and a portion comprising the appropriate soluble Jagged aminoacid residues. One skilled in the art would appreciate, based upon thedisclosure provided herein, that smaller fragments of these nucleicacids can also be used to produce antibodies that specifically bindsoluble Jagged.

One skilled in the art would appreciate, based upon the disclosureprovided herein, that various portions of an isolated soluble Jaggedpolypeptide can be used to generate antibodies to either highlyconserved regions of soluble Jagged or to non-conserved regions. Asdisclosed elsewhere herein, Jagged protein (GenBank Acc. No. U77720,[SEQ ID NO:1]), the amino acid sequence of which is depicted in FIG. 8A,comprises various conserved domains including, but not limited to, asignal peptide (from about amino acid residue 1 to about amino acidresidue 21); a DSL domain (from about amino acid residue 185 to aboutamino acid residue 229); EGF repeats (from about amino acid residue 234to about amino acid residue 862); a cysteine-rich region (from aboutamino acid residue 863 to about amino acid residue 1002); atransmembrane domain (from about amino acid residue 1068 to about aminoacid residue 1093); and a cytoplasmic region (from about amino acidresidue 1094 to about amino acid residue 1218). Once armed with thesequence of Jagged and the detailed analysis localizing the variousconserved and non-conserved domains of the protein, the skilled artisanwould understand, based upon the disclosure provided herein, how toobtain antibodies specific for the various domains using methodswell-known in the art.

Further, the skilled artisan, based upon the disclosure provided herein,would appreciate that the non-conserved regions of a protein of interestcan be more immunogenic than the highly conserved regions which areconserved among various organisms. Immunization using a non-conservedimmunogenic portion can produce antibodies specific for thenon-conserved region thereby producing antibodies that do notcross-react with other proteins which can share one or more conservedportions.

One skilled in the art would appreciate, based upon the disclosureprovided herein, which portions of soluble Jagged are less homologouswith other proteins sharing conserved domains. However, the presentinvention is not limited to any particular domain; instead, the skilledartisan would understand that other non-conserved regions of the solubleJagged proteins of the invention can be used to produce the antibodiesof the invention as disclosed herein.

The invention should not be construed as being limited solely to theantibodies disclosed herein or to any particular immunogenic portion ofthe proteins of the invention. Rather, the invention should be construedto include other antibodies, as that term is defined elsewhere herein,to soluble Jagged, or portions thereof, or to proteins sharing at leastabout 65% homology with these proteins.

The invention encompasses polyclonal, monoclonal, synthetic antibodies,and the like. One skilled in the art would understand, based upon thedisclosure provided herein, that the crucial feature of the antibody ofthe invention is that the antibody bind specifically with solubleJagged. That is, the antibody of the invention recognizes solubleJagged, or a fragment thereof, on Western blots, in immunostaining ofcells, and immunoprecipitates soluble Jagged using standard methodswell-known in the art.

One skilled in the art would appreciate, based upon the disclosureprovided herein, that the antibodies can be used to localize therelevant protein in a cell and to study the role(s) of the antigenrecognized thereby in cell processes. Moreover, the antibodies can beused to detect and or measure the amount of protein present in abiological sample using well-known methods such as, but not limited to,Western blotting and enzyme-linked immunosorbent assay (ELISA).Moreover, the antibodies can be used to immunoprecipitate and/orimmuno-affinity purify their cognate antigen using methods well-known inthe art.

The generation of polyclonal antibodies is accomplished by inoculatingthe desired animal with the antigen and isolating antibodies whichspecifically bind the antigen therefrom using standard antibodyproduction methods such as those described in, for example, Harlow etal. (1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor,N.Y.).

Monoclonal antibodies directed against full length or peptide fragmentsof a protein or peptide can be prepared using any well known monoclonalantibody preparation procedures, such as those described, for example,in Harlow et al., 1988, supra, and in Tuszynski et al. (1988, Blood,72:109-115), and methods set forth elsewhere herein. Quantities of thedesired peptide may also be synthesized using chemical synthesistechnology. Alternatively, DNA encoding the desired peptide may becloned and expressed from an appropriate promoter sequence in cellssuitable for the generation of large quantities of peptide. Monoclonalantibodies directed against the peptide are generated from miceimmunized with the peptide using standard procedures as referencedherein.

A nucleic acid encoding the monoclonal antibody obtained using theprocedures described herein may be cloned and sequenced using technologywhich is available in the art, and is described, for example, in Wrightet al. (1992, Critical Rev. Immunol. 12:125-168), and the referencescited therein. Further, the antibody of the invention may be “humanized”using the technology described in Wright et al. (supra), and in thereferences cited therein, and in Gu et al. (1997, Thrombosis andHematocyst 77:755-759).

To generate a phage antibody library, a cDNA library is first obtainedfrom mRNA which is isolated from cells, e.g., the hybridoma, whichexpress the desired protein to be expressed on the phage surface, e.g.,the desired antibody. cDNA copies of the mRNA are produced using reversetranscriptase. cDNA which specifies immunoglobulin fragments areobtained by PCR and the resulting DNA is cloned into a suitablebacteriophage vector to generate a bacteriophage DNA library comprisingDNA specifying immunoglobulin genes. The procedures for making abacteriophage library comprising heterologous DNA are well known in theart and are described, for example, in Sambrook et al., supra.

Bacteriophage which encode the desired antibody, may be engineered suchthat the protein is displayed on the surface thereof in such a mannerthat it is available for binding to its corresponding binding protein,e.g., the antigen against which the antibody is directed. Thus, whenbacteriophage which express a specific antibody are incubated in thepresence of a cell which expresses the corresponding antigen, thebacteriophage will bind to the cell. Bacteriophage which do not expressthe antibody will not bind to the cell. Such panning techniques are wellknown in the art and are described for example, in Wright et al.(supra).

Processes such as those described above, have been developed for theproduction of human antibodies using M13 bacteriophage display (Burtonet al., 1994, Adv. Immunol. 57:191-280). Essentially, a cDNA library isgenerated from mRNA obtained from a population of antibody-producingcells. The mRNA encodes rearranged immunoglobulin genes and thus, thecDNA encodes the same. Amplified cDNA is cloned into M13 expressionvectors creating a library of phage which express human Fab fragments ontheir surface. Phage which display the antibody of interest are selectedby antigen binding and are propagated in bacteria to produce solublehuman Fab immunoglobulin. Thus, in contrast to conventional monoclonalantibody synthesis, this procedure immortalizes DNA encoding humanimmunoglobulin rather than cells which express human immunoglobulin.

The procedures presented herein describe the generation of phage whichencode the Fab portion of an antibody molecule. However, the inventionshould not be construed to be limited solely to the generation of phageencoding Fab antibodies. Rather, phage which encode single chainantibodies (scFv/phage antibody libraries) are also included in theinvention. Fab molecules comprise the entire Ig light chain, that is,they comprise both the variable and constant region of the light chain,but include only the variable region and first constant region domain(CH1) of the heavy chain. Single chain antibody molecules comprise asingle chain of protein comprising the Ig Fv fragment. An Ig Fv fragmentincludes only the variable regions of the heavy and light chains of theantibody, having no constant region contained therein. Phage librariescomprising scFv DNA may be generated following the procedures describedin Marks et al. (1991, J. Mol. Biol. 222:581-597). Panning of phage sogenerated for the isolation of a desired antibody is conducted in amanner similar to that described for phage libraries comprising Fab DNA.

The invention should also be construed to include synthetic phagedisplay libraries in which the heavy and light chain variable regionsmay be synthesized such that they include nearly all possiblespecificities (Barbas, 1995, Nature Medicine 1:837-839; de Kruif et al.1995, J. Mol. Biol. 248:97-105).

Compositions

The invention includes a composition comprising an isolated purifiedsoluble Jagged, or fragment thereof, and a composition comprising anisolated nucleic acid encoding soluble Jagged. The compositions can beused, for example, to assess the level of expression of soluble Jagged,to affect the level of soluble Jagged in a cell and/or in a mammal, aswell as to affect angiogenesis, differentiation, or both, in a celland/or in a mammal, to identify useful compounds, and the like.

The invention includes a composition comprising an isolated purifiedpolypeptide comprising a soluble Jagged. Preferably, the compositioncomprises a pharmaceutically acceptable carrier. The composition can beadministered to a mammal afflicted with a disease, disorder or conditionassociated with a reduced level of soluble Jagged compared with thelevel of soluble Jagged in an otherwise identical mammal not sufferingfrom such disease, disorder or condition.

Additionally, a composition comprising an isolated purified polypeptidecomprising a soluble Jagged, or an immunogenic portion thereof, can beadministered to an animal to induce an immune response thereto. Oneskilled in the art would appreciate, based upon the disclosure providedherein, that the composition can be used to produce useful antibodiesthat specifically bind with soluble Jagged.

The invention further includes a composition comprising an isolatedsoluble Jagged, or a fragment thereof wherein the fragment comprisesamino acid residues from about 1 to about 1067 (SEQ ID NO:18) (FIG. 13A)of the full-length Jagged-1 protein (SEQ ID NO:1) depicted in FIG. 8A.

Administering soluble Jagged is useful since previous studiesdemonstrate that soluble Jagged plays a crucial role in angiogenesis(see, e.g., studies demonstrating that soluble Jagged inducesangiogenesis in a CAM assay). Thus, one skilled in the art wouldunderstand, based upon the disclosure provided herein, thatadministration of soluble Jagged is an important potential therapeuticfor treatment of a disease, disorder or condition mediated by decreasedsoluble Jagged expression, function, or both.

The invention further includes administering soluble Jagged byadministering a nucleic acid encoding soluble Jagged (e.g., a nucleicacid having at least about 20% homology with SEQ ID NO:17 whichcomprises from about nucleotide 1 to about nucleotide 3201 of SEQ IDNO:2). As more fully set forth elsewhere herein, one skilled in the artwould appreciate, based upon the disclosure provided herein, that aprotein can be administered to a cell and/or to a mammal, byadministering a nucleic acid encoding the protein. Such methods ofadministering a protein of interest, i.e., a soluble Jagged or afragment thereof, are encompassed in the present invention.

For administration of the above-mentioned compositions to a mammal, apolypeptide, or the nucleic acid encoding it, or both, can be suspendedin any pharmaceutically acceptable carrier, for example, HEPES bufferedsaline at a pH of about 7.8. Other pharmaceutically acceptable carrierswhich are useful include, but are not limited to, glycerol, water,saline, ethanol and other pharmaceutically acceptable salt solutionssuch as phosphates and salts of organic acids. Examples of these andother pharmaceutically acceptable carriers are described in Remington'sPharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides.

Pharmaceutical compositions that are useful in the methods of theinvention may be administered, prepared, packaged, and/or sold informulations suitable for oral, rectal, vaginal, parenteral, topical,pulmonary, intranasal, buccal, ophthalmic, or another route ofadministration. Other contemplated formulations include projectednanoparticles, liposomal preparations, resealed erythrocytes containingthe active ingredient, and immunologically-based formulations.

The compositions of the invention may be administered via numerousroutes, including, but not limited to, oral, rectal, vaginal,parenteral, topical, pulmonary, intranasal, buccal, or ophthalmicadministration routes. The route(s) of administration will be readilyapparent to the skilled artisan and will depend upon any number offactors including the type and severity of the disease being treated,the type and age of the veterinary or human patient being treated, andthe like.

Pharmaceutical compositions that are useful in the methods of theinvention may be administered systemically in oral solid formulations,ophthalmic, suppository, aerosol, topical or other similar formulations.In addition to the compound such as heparan sulfate, or a biologicalequivalent thereof, such pharmaceutical compositions may containpharmaceutically-acceptable carriers and other ingredients known toenhance and facilitate drug administration. Other possible formulations,such as nanoparticles, liposomes, resealed erythrocytes, andimmunologically based systems may also be used to administer solubleJagged, alone or in combination with a nucleic acid encoding the same.

The invention encompasses the preparation and use of pharmaceuticalcompositions comprising a compound useful for treatment of any disease,disorder or condition associated with altered expression of solubleJagged in a manmal. Such a pharmaceutical composition may consist of theactive ingredient alone, in a form suitable for administration to asubject, or the pharmaceutical composition may comprise the activeingredient and one or more pharmaceutically acceptable carriers, one ormore additional ingredients, or some combination of these. The activeingredient may be present in the pharmaceutical composition in the formof a physiologically acceptable ester or salt, such as in combinationwith a physiologically acceptable cation or anion, as is well known inthe art.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as cattle, pigs, horses, sheep, cats, and dogs. Inaddition, the administration of the compositions to birds is alsocontemplated.

Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal,buccal, ophthalmic, intrathecal or another route of administration.Other contemplated formulations include projected nanoparticles,liposomal preparations, resealed erythrocytes containing the activeingredient, and immunologically-based formulations.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is a discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

In addition to the active ingredient, a pharmaceutical composition ofthe invention may fturther comprise one or more additionalpharmaceutically active agents. Particularly contemplated additionalagents include anti-emetics and scavengers such as cyanide and cyanatescavengers.

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.

A formulation of a pharmaceutical composition of the invention suitablefor oral administration may be prepared, packaged, or sold in the formof a discrete solid dose unit including, but not limited to, a tablet, ahard or soft capsule, a cachet, a troche, or a lozenge, each containinga predetermined amount of the active ingredient. Other formulationssuitable for oral administration include, but are not limited to, apowdered or granular formulation, an aqueous or oily suspension, anaqueous or oily solution, or an emulsion.

As used herein, an “oily” liquid is one which comprises acarbon-containing liquid molecule and which exhibits a less polarcharacter than water.

A tablet comprising the active ingredient may, for example, be made bycompressing or molding the active ingredient, optionally with one ormore additional ingredients. Compressed tablets may be prepared bycompressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets may be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. Pharmaceutically acceptable excipients used in themanufacture of tablets include, but are not limited to, inert diluents,granulating and disintegrating agents, binding agents, and lubricatingagents. Known dispersing agents include, but are not limited to, potatostarch and sodium starch glycollate. Known surface active agentsinclude, but are not limited to, sodium lauryl sulfate. Known diluentsinclude, but are not limited to, calcium carbonate, sodium carbonate,lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include, but are not limited to, corn starch and alginic acid.Known binding agents include, but are not limited to, gelatin, acacia,pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxy propylmethyl cellulose. Known lubricating agents include, but are not limitedto, magnesium stearate, stearic acid, silica, and talc.

Tablets may be non-coated or they may be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of asubject, thereby providing sustained release and absorption of theactive ingredient. By way of example, a material such as glycerylmonostearate or glyceryl distearate may be used to coat tablets. Furtherby way of example, tablets may be coated using methods described in U.S.Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to formosmotically-controlled release tablets. Tablets may further comprise asweetening agent, a flavoring agent, a coloring agent, a preservative,or some combination of these in order to provide a pharmaceuticallyelegant and palatable preparation.

Hard capsules comprising the active ingredient may be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and may further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient may be made usinga physiologically degradable composition, such as gelatin. Such softcapsules comprise the active ingredient, which may be mixed with wateror an oil medium such as peanut oil, liquid paraffin, or olive oil.

Liquid formulations of a pharmaceutical composition of the inventionwhich are suitable for oral administration may be prepared, packaged,and sold either in liquid form or in the form of a dry product intendedfor reconstitution with water or another suitable vehicle prior to use.

Liquid suspensions may be prepared using conventional methods to achievesuspension of the active ingredient in an aqueous or oily vehicle.Aqueous vehicles include, for example, water and isotonic saline. Oilyvehicles include, for example, almond oil, oily esters, ethyl alcohol,vegetable oils such as arachis, olive, sesame, or coconut oil,fractionated vegetable oils, and mineral oils such as liquid paraffin.Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent. Known suspendingagents include, but are not limited to, sorbitol syrup, hydrogenatededible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gumacacia, and cellulose derivatives such as sodium carboxymethylcellulose,methyl cellulose, hydroxypropylmethylcellulose. Known dispersing orwetting agents include, but are not limited to, naturally-occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.,polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan monooleate,respectively). Known emulsifying agents include, but are not limited to,lecithin and acacia. Known preservatives include, but are not limitedto, methyl, ethyl, or n-propyl-para-hydroxybenzoates, ascorbic acid, andsorbic acid. Known sweetening agents include, for example, glycerol,propylene glycol, sorbitol, sucrose, and saccharin. Known thickeningagents for oily suspensions include, for example, beeswax, hardparaffin, and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily solventsmay be prepared in substantially the same manner as liquid suspensions,the primary difference being that the active ingredient is dissolved,rather than suspended in the solvent. Liquid solutions of thepharmaceutical composition of the invention may comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention may be prepared using known methods. Such formulations maybe administered directly to a subject, used, for example, to formtablets, to fill capsules, or to prepare an aqueous or oily suspensionor solution by addition of an aqueous or oily vehicle thereto. Each ofthese formulations may further comprise one or more of a dispersing orwetting agent, a suspending agent, and a preservative. Additionalexcipients, such as fillers and sweetening, flavoring, or coloringagents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared,packaged, or sold in the form of an oil-in-water emulsion or awater-in-oil emulsion. The oily phase may be a vegetable oil such asolive or arachis oil, a mineral oil such as liquid paraffin, or acombination of these. Such compositions may further comprise one or moreemulsifying agents such as naturally occurring gums such as gum acaciaor gum tragacanth, naturally-occurring phosphatides such as soybean orlecithin phosphatide, esters or partial esters derived from combinationsof fatty acids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions may also containadditional ingredients including, for example, sweetening or flavoringagents.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for rectal administration. Such acomposition may be in the form of, for example, a suppository, aretention enema preparation, and a solution for rectal or colonicirrigation.

Suppository formulations may be made by combining the active ingredientwith a non-irritating pharmaceutically acceptable excipient which issolid at ordinary room temperature (i.e., about 20° C.) and which isliquid at the rectal temperature of the subject (i.e., about 37° C. in ahealthy human). Suitable pharmaceutically acceptable excipients include,but are not limited to, cocoa butter, polyethylene glycols, and variousglycerides. Suppository formulations may further comprise variousadditional ingredients including, but not limited to, antioxidants andpreservatives.

Retention enema preparations or solutions for rectal or colonicirrigation may be made by combining the active ingredient with apharmaceutically acceptable liquid carrier. As is well known in the art,enema preparations may be administered using, and may be packagedwithin, a delivery device adapted to the rectal anatomy of the subject.Enema preparations may further comprise various additional ingredientsincluding, but not limited to, antioxidants and preservatives.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for vaginal administration. Such acomposition may be in the form of, for example, a suppository, animpregnated or coated vaginally-insertable material such as a tampon, adouche preparation, or gel or cream or a solution for vaginalirrigation.

Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include, but are not limited tomethods of depositing or binding a chemical composition onto a surface,methods of incorporating a chemical composition into the structure of amaterial during the synthesis of the material (i.e., such as with aphysiologically degradable material), and methods of absorbing anaqueous or oily solution or suspension into an absorbent material, withor without subsequent drying.

Douche preparations or solutions for vaginal irrigation may be made bycombining the active ingredient with a pharmaceutically acceptableliquid carrier. As is well known in the art, douche preparations may beadministered using, and may be packaged within, a delivery deviceadapted to the vaginal anatomy of the subject. Douche preparations mayfurther comprise various additional ingredients including, but notlimited to, antioxidants, antibiotics, antifungal agents, andpreservatives.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intraperitoneal, intramuscular, intrasternal injection, and kidneydialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e., powder or granular) form for reconstitution witha suitable vehicle (e.g., sterile pyrogen-free water) prior toparenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulationswhich are useful include those which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer systems. Compositions for sustained release orimplantation may comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

Formulations suitable for topical administration include, but are notlimited to, liquid or semi-liquid preparations such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for pulmonary administration via thebuccal cavity. Such a formulation may comprise dry particles whichcomprise the active ingredient and which have a diameter in the rangefrom about 0.5 to about 7 nanometers, and preferably from about 1 toabout 6 nanometers. Such compositions are conveniently in the form ofdry powders for administration using a device comprising a dry powderreservoir to which a stream of propellant may be directed to dispersethe powder or using a self-propelling solvent/powder-dispensingcontainer such as a device comprising the active ingredient dissolved orsuspended in a low-boiling propellant in a sealed container. Preferably,such powders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers. Morepreferably, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositionspreferably include a solid fine powder diluent such as sugar and areconveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally, thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic or solid anionic surfactant or a solid diluent(preferably having a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may also provide the active ingredient in the form of dropletsof a solution or suspension. Such formulations may be prepared,packaged, or sold as aqueous or dilute alcoholic solutions orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization or atomizationdevice. Such formulations may further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, or a preservative such as methylhydroxybenzoate. The dropletsprovided by this route of administration preferably have an averagediameter in the range from about 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary deliveryare also useful for intranasal delivery of a pharmaceutical compositionof the invention.

Another formulation suitable for intranasal administration is a coarsepowder comprising the active ingredient and having an average particlefrom about 0.2 to 500 micrometers. Such a formulation is administered inthe manner in which snuff is taken, i.e., by rapid inhalation throughthe nasal passage from a container of the powder held close to thenares.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) to as much as 100% (w/w) ofthe active ingredient, and may further comprise one or more of theadditional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets or lozengesmade using conventional methods, and may, for example, comprise fromabout 0.1 to 20% (w/w) active ingredient, the balance comprising anorally dissolvable or degradable composition and, optionally, one ormore of the additional ingredients described herein. Alternately,formulations suitable for buccal administration may comprise a powder oran aerosolized or atomized solution or suspension comprising the activeingredient. Such powdered, aerosolized, or aerosolized formulations,when dispersed, preferably have an average particle or droplet size inthe range from about 0.1 to about 200 nanometers, and may furthercomprise one or more of the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for ophthalmic administration. Suchformulations may, for example, be in the form of eye drops including,for example, a 0.1-1.0% (w/w) solution or suspension of the activeingredient in an aqueous or oily liquid carrier. Such drops may furthercomprise buffering agents, salts, or one or more other of the additionalingredients described herein. Other ophthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form or in a liposomal preparation.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed. (1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa.), which isincorporated herein by reference.

Typically dosages of the compound of the invention which may beadministered to an animal, preferably a human, range in amount from 1microgram to about 100 grams per kilogram of body weight of the animal.While the precise dosage administered will vary depending upon anynumber of factors, including but not limited to, the type of animal andtype of disease state being treated, the age of the animal and the routeof administration. Preferably, the dosage of the compound will vary fromabout 1 milligram to about 10 grams per kilogram of body weight of theanimal. More preferably, the dosage will vary from about 10 milligramsto about 1 gram per kilogram of body weight of the animal.

The compound may be administered to an animal as frequently as severaltimes daily, or it may be administered less frequently, such as once aday, once a week, once every two weeks, once a month, or even lessfrequently, such as once every several months or even once a year orless. The frequency of the dose will be readily apparent to the skilledartisan and will depend upon any number of factors, such as, but notlimited to, the type and severity of the disease being treated, the typeand age of the animal, etc.

Methods

The present invention also includes a method of affecting angiogenesisin a system capable of angiogenesis. As more fully set forth elsewhereherein in discussing methods of using a Jagged protein or a functionallyequivalent derivative or allelic or species variant thereof, a solubleJagged protein can be used to affect angiogenesis due to the role of theJagged-Notch signaling pathway in angiogenesis. That is, the datadisclosed herein demonstrate that contacting certain cells with solubleJagged mediates angiogenesis in “systems capable of angiogenesis,” asexemplified by formation of tissue masses in nude mice, sprout formationby endothelial cells, cell migration, and angiogenesis detected in a CAMassay. One skilled in the art would appreciate, based upon thedisclosure provided herein, that there are numerous systems capable ofangiogenesis under the proper conditions where angiogenesis can beassessed such as those disclosed herein, as well as systems well-knownin the art and those to be developed in the future, all of which areencompassed in the present invention.

More specifically, in one embodiment, transfected cells expressingsoluble Jagged demonstrated altered growth in culture and/or formationof tissue masses in nude mice and/or angiogenic potential in CAM assaycompared to a FGF-2 positive control. As stated previously elsewhereherein, one skilled in the art would appreciate, based upon thedisclosure provided herein, that the ability of a soluble Jagged proteinto affect angiogenesis can be measured not only by the aforementionedassays but by any similar assay now available or which is developed inthe future to measure angiogenic potential.

Further, one skilled in the art would appreciate, based upon the instantdisclosure, that angiogenesis can be affected not only by the additionof exogenous soluble Jagged protein, but can also be affected by theintroduction of an exogenous nucleic acid encoding soluble Jagged into acell where it is expressed, and/or by the introduction into a mammal ofcells which express the protein which is encoded by a soluble Jaggednucleic acid. Thus, the method of the present invention is not limitedto any particular manner in which the Jagged protein and/or solubleJagged is provided to a cell or to a mammal; rather, the inventionencompasses various methods whereby a Jagged protein, a soluble Jagged,and/or a portion thereof, is introduced to a cell or into a mammal.

As more fully set forth elsewhere herein, a soluble Jagged protein canbe administered to a mammal via a variety of routes. Further, the dosageand amounts administered depend on numerous factors which are discussedmore fully elsewhere herein in.

The amount of soluble Jagged administered, whether it is administered asprotein or as nucleic acid or as a cell expressing soluble Jagged, issufficient to elicit a Jagged/Notch signaling response. Thepharmaceutical compositions useful for practicing the invention can beadministered to deliver a dose of between about 1 nanogram per kilogramand about 100 milligrams per kilogram of soluble Jagged protein perpatient body weight. Suitable amounts of the soluble Jagged protein foradministration include doses which are high enough to have the desiredeffect without concomitant adverse effects. When the soluble Jagged is aprotein or peptide, a preferred dosage range is from about 1 pg to about100 mg of protein or peptide per kg of patient body weight.

When the soluble Jagged is administered in the form of DNA encoding thesame contained within a recombinant virus vector, a dosage of betweenabout 10² and about 10¹¹ plaque forming units of virus per kilogram ofpatient body weight can be used. When naked DNA encoding the solubleJagged is to be administered as the pharmaceutical composition, a dosageof between about 1 pg to about 100 mg of DNA per kilogram of patientbody weight can be used. Further, when the soluble Jagged isadministered in the form of a cell expressing a nucleic acid encodingthe same, the dosage of cells per kilogram of patient body weight can beassessed depending on the amount of soluble Jagged protein expressed bythe cells and the level desired as disclosed previously elsewhereherein.

When soluble Jagged is administered by administering a nucleic acidencoding the protein, the nucleic acid can be administered naked (e.g.,substantially free of any other substance with which a nucleic acid istypically associated such as protein, and the like). Alternatively, thenucleic acid can be encapsulated or otherwise associated with anothersubstance capable of facilitating the introduction of the nucleic acidinto a cell. Such nucleic acid delivery techniques are describedelsewhere herein and are well-known in the art and are described in, forexample, Sambrook et al., supra, and Ausubel et al., supra.

An angiogenic effective amount, as that term is used and definedelsewhere herein, can be readily determined using any of theangiogenesis assays disclosed herein as well as methods well-known inthe art. That is, the angiogenic effect of a soluble Jagged administeredto a cell and/or to an organism or assay system, can be assessed by, forexample, measuring the effect of soluble Jagged on expression of variousgenes (e.g., using differential display analyses such as SAGE analysis),migration of cells in culture, formation of chords by cells grown onplastic or on collagen matrices, assessing the level of repression oftype I collagen expression, measuring the angiogenic potential using aCAM assay and/or measuring the in vivo growth of the cell usingtransplant studies in various murine models. However, the presentinvention is not limited to these assays to detect effects of solubleJagged on angiogenesis; rather, similar assays which are now known orwhich are developed in the future may be used to determine the effect ofsoluble Jagged protein on angiogenesis.

The invention also includes a method of affecting differentiation of acell. The method comprises contacting a cell with an effective amount ofa substantially purified soluble Jagged protein. One skilled in the artwould appreciate, based upon the disclosure provided herein, thatcontacting a cell with a soluble Jagged protein mediates signaling viathe Jagged/Notch pathway such that cell differentiation, angiogenesis,and other cellular processes, are affected as demonstrated by the datadisclosed herein.

One skilled in the art would further appreciate, based upon thedisclosure provided herein, that a cell whose differentiation can beaffected by contacting the cell with soluble Jagged should express aJagged receptor, e.g., Notch, and comprise all necessary components ofthe Jagged/Notch signaling pathway such that Jagged/Notch interactionsinvolved in differentiation can be affected by contacting the cell withsoluble Jagged. Cells that express a Jagged receptor that can be usedfor such an assay include, but are not limited to, anymesodermal-derived cell, any endodermal-derived cell, anyectodermal-derived cell, and any neurodermal-derived cell, and the like.In addition to cells that naturally express an endogenous Jaggedreceptor, the present invention encompasses cells that have beenmanipulated such that they express a Jagged receptor and comprise thenecessary Jagged/Notch signaling pathway so that the effect of solubleJagged upon differentiation can be assessed in the cell.

A differentiation effective amount, as that term is defined elsewhereherein, of soluble Jagged protein can be readily determined by assessingthe effect(s) of contacting a cell with soluble Jagged or a fragmentthereof. Such methods include, but are not limited to, those disclosedherein which include measuring the effect of soluble Jagged onexpression of various genes (e.g., using differential display analysessuch as SAGE analysis) including repression of type I collagenexpression, growth of cells on plastic or on collagen matrices,formation of chords and/or tubes by cells grown on plastic or oncollagen matrices, measuring the angiogenic potential using a CAM assayand/or measuring the in vivo growth of the cell using transplant studiesin various murine models. However, the present invention is not limitedto these assays to detect effects of soluble Jagged on celldifferentiation; rather, similar assays which are now known or which aredeveloped in the future may be used to determine the effect of solubleJagged protein on differentiation.

Further, the invention includes a method of identifying a compoundcapable of affecting differentiation in a cell. The method comprisescontacting a soluble Jagged transfectant cell with a test compound andcomparing the growth characteristics of the cell with the growthcharacteristics of an otherwise identical soluble Jagged transfectantcell not contacted with the compound. One skilled in the art wouldappreciate, based upon the disclosure provided herein, that comparingthe growth characteristics of a soluble Jagged transfectant cell, whichis/are an indicator of differentiation, allows the identification of acompound that affects cell differentiation.

By the term “growth characteristics,” as the term is used herein, ismeant any change in growth kinetics, size, morphology, and/orassociation with other cells exhibited by a cell transfected withnucleic acid encoding a soluble Jagged which is not exhibited by anidentical cell which is not transfected or which is transfected with anempty, insert-less vector. As disclosed herein, such growthcharacteristics include, but are not limited to, the ability to formchord-like structures (shown in FIG. 10) when grown in vitro; theability to form tissue masses when transplanted into nude mice (as shownin FIG. 12); and the ability to form angiogenic structures in CAMassays.

Further, the growth characteristics include the pattern of geneexpression as assessed using, for example, a modified differentialdisplay method such as serial analysis of gene expression (SAGE)analysis as exemplified herein. This is because, as stated previouslyelsewhere herein, the pattern of gene expression is correlated to celldifferentiation such that changes in the pattern are indicative ofdifferentiation in the cell. Thus, the pattern of gene expression in thecell contacted with a test compound can be compared to the pattern in anotherwise identical cell not contacted with the compound and/or with thepattern in the cell prior to being contacted with the compound. Thealtered level of expression in certain genes can be assessed and used todetect differentiation in a cell since the data disclosed hereindemonstrate that soluble Jagged-mediated differentiation causes thelevel of certain transcripts to decrease while causing the level ofother transcripts to increase. Therefore, changes in the pattern of geneexpression in a cell can be used to indicate differentiation in the cellmediated by soluble Jagged and the effect(s) of a test compound on suchdifferentiation.

However, the present invention should not be construed to be limited tothese or any other particular growth characteristics or assays todetermine cell differentiation. Rather, any growth characteristicdemonstrated by a cell transfected with a nucleic acid encoding asoluble Jagged which is not exhibited by an otherwise identical cell nottransfected, or transfected with an empty vector, may be used inidentifying a test compound capable of affecting cell differentiation.This is because, as will be appreciated by one skilled in the art basedupon the disclosure provided herein, a growth characteristic exhibitedby a soluble Jagged transfectant but not exhibited by an otherwiseidentical cell which is not transfected (or which is transfected by anempty, insert-less vector) is due, at least in part, by the alteredJagged/Notch signaling in the transfectant and the Jagged/Notchsignaling pathway is known to be involved in cell differentiation,angiogenesis, and the like. Thus, a compound that affects a growthcharacteristic mediated by the Jagged/Notch signaling pathway affectscell differentiation since differentiation is also mediated by suchpathway.

Similarly, the present invention includes a method of identifying acompound capable of affecting the binding of Jagged ligand to a Notchreceptor. The method comprises contacting a soluble Jagged-transfectedcell with a test compound and comparing the growth characteristics ofthe cell contacted with the compound with the growth characteristics ofan otherwise identical cell not contacted with the compound. Asdiscussed previously herein, a difference in the growthcharacteristic(s), including any change in the pattern of geneexpression otherwise mediated by soluble Jagged, of the transfectantcell contacted with the compound compared with the growthcharacteristic(s) of the otherwise identical transfectant cell notcontacted with the compound is an indication that the compound iscapable of affecting the binding of Jagged ligand to a Notch receptor.This is because the growth characteristic(s) is the result of thealtered Jagged/Notch signaling pathway present in the soluble Jaggedtransfectant cell which, if affected by a substance, indicates that thesubstance affects Jagged/Notch binding. Therefore, as will beappreciated by one skilled in the art based upon the disclosure providedherein, a change in a growth characteristic associated with or mediatedby the altered Jagged/Notch signaling pathway upon contact with a testcompound is an indication of the ability of the test compound to affectsuch pathway, and, therefore, to affect Jagged/Notch binding.

The invention also includes a method of identifying a compound capableof affecting angiogenesis. The method comprises contacting a solubleJagged transfectant cell with a test compound and comparing the growthcharacteristics of the cell contacted with the compound with the growthcharacteristics of an otherwise identical cell not contacted with thecompound. As discussed previously herein, a difference in the growthcharacteristic(s), including any change in the pattern of geneexpression otherwise mediated by soluble Jagged, of the transfectantcell contacted with the compound compared with the growthcharacteristic(s) of the otherwise identical transfectant cell notcontacted with the compound is an indication that the compound iscapable of affecting angiogenesis. This is because the growthcharacteristic(s) present in the soluble Jagged transfectant cell, isthe result of the altered Jagged/Notch signaling pathway and theJagged/Notch signaling pathway mediates angiogenesis. Thus, the growthcharacteristic(s) in the transfected cell mediated by soluble Jagged areinvolved in angiogenesis such that if the growth characteristic(s)is/are affected by a substance, such response indicates that thesubstance affects angiogenesis.

Therefore, as will be appreciated by one skilled in the art based uponthe disclosure provided herein, a change in a growth characteristicassociated with or mediated by the altered Jagged/Notch signalingpathway upon contact with a test compound is an indication of theability of the test compound to affect such pathway, and, therefore, toaffect angiogenesis mediated by Jagged/Notch signaling.

The present invention further provides methods of regulating geneexpression in a cell. For example, a cell can be altered such that itcontains a DNA sequence operably linked to a RE. Additionally, the cellcan be altered to control the expression of Jagged permitting oneskilled in the art to generate a cell which expresses a given sequencein response to a particular agent.

The subjects treated in accordance with the present invention includeany vertebrate organism; more preferably any mammal; most preferably ahuman. The only limiting factor is that the organism endogenouslyproduces Notch and/or the toporythmic genes which modulate binding toNotch.

By providing methods of affecting angiogenesis by modulating theNotch-Jagged signal pathway, the present invention provides methods andcompositions which affect a number of physiologic and pathologicconditions, including placental development, wound healing, rheumatoidarthritis, diabetic retinopathy and solid tumor growth and metastasisand motor neuron disorders. The referenced wound healing includeshealing of any injury or lesion in the skin, tissue, vasculature, ornervous system of the subject, and includes cell migration anddifferentiation of cells comprising the mesoderm, endoderm, ectodermand/or neuroderm. The wound or injury can be the result of surgery,trauma, and/or disease or condition. Such disease and/or conditionsinclude ischemic lesions resulting from a lack of oxygen to the cell ortissue, e.g., cerebral or cardiac infarction or ischemia, malignantlesions, infectious lesions, e.g., abscess, degenerative lesions,lesions related to nutritional disorders, neurological lesionsassociated with systemic diseases, e.g., diabetic neuropathy andretinopathy, systemic lupus erythematosus, carcinoma or sarcoidosis, andlesions caused by toxins, e.g., alcohol, lead, etc. Motor neurondisorders may include, e.g., amylotrophic lateral sclerosis, progressivespinal muscular atrophy, progressive bulbar palsy, primary lateralsclerosis, infantile and juvenile muscular atrophy, progressive bulbarparalysis of childhood (Fazio-Londe syndrome), poliomyelitis and thepost polio syndrome, and hereditary Motorsensory Neuropathy(Charcot-Marie-Tooth disease).

The invention also includes a method of inhibiting expression of type Icollagen in a cell. The method comprises administering an expressioninhibiting amount of soluble Jagged to a cell, thereby inhibitingexpression of type I collagen. One skilled in the art would understandthat various type I collagens (e.g., pro-α-1(I) collagen, pro-α-2(I)collagen, and the like) are encompassed by the invention which is notlimited to any particular type I collagen.

One skilled in the art would also appreciate, based upon the disclosureprovided herein, that soluble Jagged-1 can be administered to a cell viaa variety of methods including, but not limited to, administering anucleic acid encoding soluble Jagged, a vector encoding soluble Jagged,and an isolated soluble Jagged. The important feature is not how thesoluble Jagged is delivered to the cell but, rather, that soluble Jaggedbe administered to the cell in sufficient quantity to affectJagged/Notch interactions involved in Jagged/Notch signaling so as torepress expression of a type I collagen gene.

The level of soluble Jagged required to inhibit type I collagenexpression can be readily determined using the assays disclosed hereinor other assays well-known in the art and/or based from the assaysdisclosed elsewhere herein. For example, such assays include, but arenot limited to, assessing the level of type I collage gene expressionusing SAGE analysis and/or other nucleic-acid based assays (e.g.,Southern blotting, Northern blotting, slot-blots, PCR-based methods, andthe like). In addition, type I collagen expression can be determined byassessing the production of a specific type I collagen domain, e.g., theamino-terminal peptide portion of pro-α-1(I), and the like, usingantibody-based detection methods, which are well-known in the art and/ordisclosed elsewhere herein (e.g., immunoblotting, ELISA,immunoprecipitation, and such).

Methods of inhibiting type I collagen expression are of crucialimportance in the development of therapeutics for a plethora of fibroticdiseases associated with production of type I collagen for which thereis currently no effective treatment.

Kits

The invention includes various kits which comprise a compound, such asan isolated nucleic acid encoding soluble Jagged in a sense or in anantisense orientation, or an isolated soluble Jagged polypeptide, or theantibodies of the invention, and instructional materials which describeuse of the compound to perform the methods of the invention. Althoughexemplary kits are described below, the contents of other useful kitswill be apparent to the skilled artisan in light of the presentdisclosure. Each of these kits is included within the invention.

In one aspect, the invention includes a kit for affecting angiogenesisin a mammal. The kit is used pursuant to the methods disclosed in theinvention. Briefly, the kit may be used to introduce an isolated solubleJagged polypeptide, an isolated nucleic acid encoding soluble Jagged,and/or a cell expressing soluble Jagged into a mammal in order toincrease the level of soluble Jagged in the mammal. This affectsangiogenesis in that, as disclosed previously elsewhere herein, solubleJagged affects the Jagged/Notch signaling pathway which, in turn,affects angiogenesis. Thus, administering soluble Jagged to a mammal,either by administering soluble Jagged protein, a nucleic acid encodingsoluble Jagged, and/or a cell expressing soluble Jagged, affectsangiogenesis in the mammal.

Moreover, the kit comprises an instructional material for the use of thekit. These instructions simply embody the disclosure provided herein.

The invention further includes a kit for affecting differentiation in acell. The kit comprises an effective amount of an isolated solubleJagged polypeptide, an applicator, and an instructional material for theuse of the kit. One skilled in the art would appreciate, based upon thedisclosure provided herein, that the kit can be used to administersoluble Jagged to a cell, either by administering to such cell at leastone of the following: isolated soluble Jagged protein, and/or a nucleicacid encoding soluble Jagged that is expressed in the cell. SolubleJagged, in turn, mediates differentiation in the cell via theJagged/Notch signaling pathway. Thus, by affecting the Jagged/Notchsignaling pathway, soluble Jagged affects differentiation in a cellwhich is either contacted with soluble Jagged, or which expresses theprotein.

Moreover, the kit comprises an instructional material for the use of thekit. These instructions simply embody the disclosure provided herein.

The invention includes a kit for inhibiting expression of type Icollagen in a cell. The kit comprises an expression inhibiting amount ofsoluble Jagged, an applicator, and an instructional material for the useof said kit.

One skilled in the art would appreciate, based upon the disclosureprovided herein, that the inhibiting amount of soluble Jagged can bereadily determined using the assays disclosed herein to assess reductionof type I collagen expression (e.g., SAGE analysis, immunoblotting).Further, standard assays well-known in the art, and discussed elsewhereherein, can also be used to assess the level of soluble Jagged to beadministered and the level of type I collagen expressed, correlated withthe administration of soluble Jagged. That is, a wide plethora of assayscan be used to assess the level of type I collagen nucleic acid and/orprotein produced in a cell compared with an otherwise identical cell towhich soluble Jagged is not administered. Thus, the expressioninhibiting amount of soluble Jagged can be easily determined based uponthe disclosure provided herein.

Additionally, the invention is not limited to any particular type Icollagen; rather, the invention includes various type I collagens, e.g.,pro-α-1(I) collagen, pro-α-2(I) collagen, and the like.

Moreover, the kit comprises an instructional material for the use of thekit. These instructions simply embody the disclosure provided herein.

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

All essential publications mentioned herein are hereby incorporated byreference.

EXAMPLES

In the following examples and protocols, restriction enzymes, ligase,labels, and all commercially available reagents were utilized inaccordance with the manufacturer's recommendations. The cell andmolecular methods utilized in this application are established in theart and will not be described in detail. However, standard methods andtechniques for cloning, isolation, purification, labeling, and the like,as well as the preparation of standard reagents were performedessentially in accordance with Sambrook et al. (1989, Molecular Cloning,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), and therevised third edition thereof, Ausubel et al.(1997, Current Protocols inMolecular Biology, John Wiley & Sons, New York), or as set forth in theliterature references cited and incorporated herein. Methodologicdetails may be readily derived from the cited publications.

Example 1 Isolation of Human Endothelial Cell cDNA Induced by Exposureto Fibrin

Endothelial cells plated on fibrin organize into three dimensionaltubular structures in vitro (Olander et al., 1985, J. Cell. Physiol.125:1-9), and this organizational behavior requires transcriptionalresponses (Zimrin et al., 1995). Using a modification of thedifferential display, cDNA clones were obtained that were differentiallyexpressed by HUVECs in response to fibrin. Briefly, total RNA wasisolated from HUVEC plated on fibrin in the presence of crude FGF-1 at0, 2, 5 and 24 hours and subjected to the modified differential mRNAdisplay. One of the clones (D9) isolated from HUVEC populations exposedto fibrin, which was found to have increased at the 2 hour time-point,was cloned and sequenced. A search of the GenBank database in 1994demonstrated that the D9 sequence was novel.

The D9 clone was used as a probe to screen a lambda cDNA libraryprepared from mRNA obtained from HUVECs exposed to fibrin gels for 1, 3and 5 hours. Ten isolates were recovered that contained the D9 sequence,two of which appeared, by restriction enzyme analysis, to be splicedvariants of the remaining eight. Sequence analysis of the clonesrevealed that they overlapped to form a contiguous sequence of 5454 basepairs (bp) in length, set forth as SEQ ID NO:2.

Example 2 Analysis of the Sequence of HUVEC Clone D9 DemonstratesHomology With the Rat Jagged Gene

A second search of the Genebank database in 1995 revealed that the D9clone was very homologous to the cDNA sequence coding for the rat Jaggedgene (Lindsell et al., 1995, Cell 80:909-917), a ligand for the Notchreceptor. Computer analysis revealed an 87% identity at the nucleotidelevel and a 95% identity at the amino acid level. The Jagged protein(GenBank Acc. No. U77720, FIG. 8A [SEQ ID NO:1]) contains a putativesignal sequence (from about amino acid residue 1 to about amino acidresidue 21), a DSL domain which describes a consensus region present inother Notch ligands (Delta, Serrate, Lag-2 and Apx-1) (from about aminoacid residue 185 to about amino acid residue 229), an EGF-like repeatdomain containing sixteen EGF repeats (from about amino acid residue 234to about amino acid residue 862), a cys-rich domain (from about aminoacid residue 863 to about amino acid residue 1002), a transmembranedomain (from about amino acid residue 1068 to about amino acid residue1093), and a cytosol domain (from about amino acid residue 1094 to aboutamino acid residue 1218) (see FIG. 8A). This structure is schematicallyrepresented in FIG. 2. Thus, it was determined that clone D9 representsthe human homolog of the rat Jagged cDNA.

Two additional Jagged clones were also obtained each containingidentical deletions. The first was 89 bp in length, and was located inthe middle of the cys-rich region. The second clone occurred 366 bpdownstream from the first clone, and was approximately 1307 bp inlength. The first deletion predicts a frame-shift in the translationproduct, resulting in a unique 15 amino acid sequence followed by apremature termination of the protein, effectively deleting thetransmembrane and cytosol domains from the Jagged structure. Nucleicacids encoding truncated Jagged-1 protein (termed “soluble Jagged”,“sol-jag”, or “sJ-1,” which are used interchangeably herein) were usedto produce transfected NIH 3T3 cells expressing soluble Jagged whichcells demonstrated altered angiogenic potential in both in vivotransplantation studies using nude mice and in traditional CAM assays(see Examples 8 and 9, infra).

Example 3 Human Endothelial Cell Populations Express Both Jagged andNotch Transcripts

To ascertain that both the human Jagged gene and its putative receptor,Notch, were expressed in the HUVEC population, oligonucleotide primerswere designed based upon the published sequence for the human Tan-1transcript (Notch-1) and the human Notch group protein transcript(Notch-2), as well as for the human Jagged transcript.

Total RNA was obtained using standard protocols. The differentialdisplay was performed as previously described by Folkman andHaudenschild (1980, Nature 288:551-556). Briefly, 1 μg of total RNA wasreverse transcribed with 200U M-MLV reverse transcriptase (BRL) in thepresence of 2 μM of the 3′ primer (5′-GCGCAAGCT₁₂CG-3′ [SEQ ID NO:3])and 100 μM dNTP for 70 minutes at 37° C. The cDNA was amplified in thepresence of (³²p) dATP (Amersham) using the same 3′ primer and a 5′primer with the sequence 5′-GAGACCGTGAAGATACTT-3′ (SEQ ID NO:4) and thefollowing parameters: 94° C. 45 seconds, 41° C. 1 minute, 72° C. 1minute for 4 cycles, followed by 94° C. 45 seconds, 60 ° C. 1 minute,72° C. 1 minute for 18 cycles. The resulting cDNA species were separatedusing polyacrylamide gel electrophoresis, the gel was dried and exposedto radiographic film, and the band of interest was cut out of the geland eluted.

The cDNA was amplified using the same primers and cloned into a TAvector (Invitrogen, Carlsbad, Calif.). The clone was used to screen acDNA library made in the ZAP Express vector (Stratagene, La Jolla,Calif.) using RNA isolated from HUVEC plated on fibrin in the presenceof crude FGF-1 for 1, 3, 5, 8 and 24 hours to analyze the steady-statelevels of the transcripts for Jagged, Notch 1, Notch 2, and GAPDH. SeeGarfinkel et al., 1996, J. Cell Biol. 134:783-791. The overlapping cDNAclones obtained were sequenced using an ABI DNA synthesizer andassembled with the DNASTAR program. RT-PCR analysis was performed asdescribed using the following primers:

jagged sense 5′-CCGACTGCAGAATAAACATC-3′ (SEQ ID NO:5);

jagged antisense 5′-TTGGATCTGGTTCAGCTGCT-3′ (SEQ ID NO:6);

notch 1 sense 5′-TTCAGTGACGGCCACTGTGA-3′(SEQ ID NO:7);

notch 1 antisense 5′-CACGTACATGAAGTGCAGCT-3′ (SEQ ID NO:8);

notch 2 sense 5′-TGAGTAGGCTCCATCCAGTC-3′ (SEQ ID NO:9);

notch 2 antisense 5′-TGGTGTCAGGTAGGGATGCT-3′ (SEQ ID NO:10);

GAPDH sense 5′-CCACCCATGGCAAATTCCATGGCA-3′ (SEQ ID NO:11);

GAPDH antisense 5′-TCTAGACGGCAGGTCAGGTCCACC-3′ (SEQ ID NO:12).

As shown in FIG. 5, the steady state levels of the Notch-1 and Notch-2transcripts were not altered in HUVEC populations exposed to fibrin. Incontrast, however, the HUVEC Jagged transcript was induced after threehours exposure to fibrin after which time the steady state levels of theJagged transcript decreased (FIG. 5).

Example 4 The Role of Jagged as a Mediator of Microvascular SproutFormation In Vitro

Because (i) Delta/Serrate signaling through Notch is involved in thedetermination of cell fate in invertebrates (Fortini andArtavanis-Tsakonas, 1993, Cell 75:1245-1247), (ii) Jagged signalingthrough Notch attenuates the terminal differentiation of myoblasts tomyotubes in vitro (Lindsell et al., 1995, Cell 80:909-917), (iii) theendothelial cell presents a non-terminal differentiated phenotype invitro (FIG. 1), and (iv) the Jagged transcript was identified as anendothelial cell differentiation-induced gene, it was important todetermine whether Jagged-Notch signaling in the endothelial cell wasinvolved in the early phase of the differentiation pathway. It is wellknown that endothelial cell sprout formation is an early event in themicrovasculature during angiogenesis (Montesano and Orci, 1985, Cell42:469-477); and endothelial cell sprout formation assays are describedin the art (Montesano et al., 1986, Proc. Natl. Acad. Sci. USA83:7297-7301). However, to assess the role of Jagged-Notch signaling inthis system, an antisense (γ) oligonucleotide was needed, based on theJagged sequence to repress the translation of the Jagged transcript.

The γ-Jagged oligomer contained the Kozak sequence, the ATG translationstart site and extended three codons into the open-reading frane.Similar γ-oligomers have proven useful in a wide variety of cellularsystems to repress the translation of specific transcripts, includingthe human endothelial cell (Maier et al., 1990, J. Biol. Chem.265:10805-10808; Garfinkel et al., 1992, J. Biol. Chem.267:24375-24378). The controls for the γ-Jaggged oligomer included thesense counterpart, a 3′-antisense oligomer and a mutated 5′ antisenseoligomer.

Although the complete DNA sequence of the bovine Jagged transcript hadnot yet been fully defined, a high degree of homology at the 5′ end waspredicted between the bovine and the human Jagged nucleotide sequence,in view of the fact that the human and rat Jagged polypeptides are 95%identical.

Bovine microvascular endothelial cells (BMEC) were plated onto acollagen gel, grown to confluence in the presence or absence of variedconcentrations of the γ-Jaggged oligomer. FGF-2 (10 ng/ml) was added atconfluence (Montesano et al., 1986, Proc. Natl. Acad. Sci. USA83:7297-7301), and the length of microvessels (sprouts formed as aresult of cellular invasion into the collagen gel) was measured (Pepperet al., 1992, Biochem. Biophys. Res. Comm. 189:824-831). As shown inFIG. 6, exposure to the antisense γ-Jaggged oligomer (JAS; SEQ ID NO:29)resulted in an increase in BMEC sprout length in a concentrationdependent manner above the level achieved by FGF-2. In contrast, thethree control oligomers, a Jagged sense oligonucleotide (JS; SEQ IDNO:30), a 3′ antisense Jagged oligomer (3′ AS; SEQ ID NO:31), and amutated 5′ antisense Jagged oligomer (MUT5′ AS; SEQ ID NO:32) did notaffect the ability of FGF-2 (bFGF) to induce sprout formation in thisassay (FIG. 6).

Prior to this experiment, with the possible exception of vascularendothelial cell growth factor (VEGF), no other growth factor/cytokinesignal has been disclosed as able to potentiate the ability of FGF tomodify BMEC sprout length. This result would not have been previouslyanticipated since the Jagged gene had been previously identified as aHUVEC-derived differentiation-induced transcript.

Example 5 The Disparate Effect of the Antisense (γ)-Jagged Oligomer onSmall and Large Vessel Endothelial Cell Migration

Based upon the surprising effect of the γ-Jaggged oligomers on thepotentiation of FGF-2-induced BMBC sprout formation (Example 4), asimple assay was designed to assess the influence of the γ-Jagggedoligomer on BMEC migration, specifically to confirm that interruptingthe Jagged-Notch signaling pathway would attenuate the ability of FGF toincrease sprout length. Utilizing essentially the system of Sato andRifkin (1988, supra), bovine microvascular endothelial cells (BMEC) wereplated on a fibronectin matrix, and grown to confluence in the absenceand presence of varied amounts of the γ-Jaggged oligomer.

Briefly, 4×10⁵ BMEC and BAEC were grown to confluence inserum-containing media containing 0, 1.25, 2.5, 5 and 6.25 μM Jaggedantisense oligonucleotide. The monolayers were wounded by scraping themwith a razor blade and cellular debris was removed by washing the platestwice with phosphate buffered saline. The cells were incubated for afurther 22 hours at 37 ° C. to confluence, then fixed in 25% aceticacid, 75% methanol and stained with hematoxylin (Sigma Chemical Co., St.Louis, Mo.). The number of cells migrating from the wound origin werecounted to determine the ability of the BMEC population to migrate intothe denuded area. The count was made using a light microscope with agrid at 100×magnification. The data represent a mean of multipleexperiments done in duplicate, with five microscopic fields counted foreach point.

As shown in FIG. 7A, the presence of the γ-Jaggged oligomer resulted inan increase in the number of cells migrating into the denuded area withan approximate 80% increase mediated by 5 μM γ-Jaggged oligomer. Thesedata (FIG. 7A) agree with the BMEC data obtained from the sprout assayin which 2 μM γ-Jaggged oligomer yielded an approximate 100% increase inBMEC sprout length (FIG. 6). Thus, it was shown that an interruption inthe Jagged-Notch signaling pathway resulted in an increase in BMECmigration, a major immediate-early component of sprout formation invitro.

Consequently, an apparent discrepancy was noted between the results ofthe experiments showing (i) the isolation of the Jagged transcript froma HUVEC population preparing to migrate into a fibrin gel, and (ii) theenhancement of the BMEC by the presumed interruption of the Jaggedsignal. Noting that the HUVEC are obtained from a macro-vessel, and BMECare from micro-vessels, the distinction was apparently directly relatedto the nature of the source of the endothelial cells.

To ascertain that the difference was based upon the type of theendothelial cell (macro- versus micro-vasculature), and not due tovariations in the extracellular matrix or the function of growthfactors/cytokines in the particular system, an experiment was designedin which the endothelial cells were obtained from the same species, butexclusively from a macrovascular source—bovine aorta endothelial cells(BAEC). BAEC were introduced onto a fibronectin matrix, grown toconfluence in the absence and presence of various amounts of theγ-Jaggged oligomer, and their migration assessed in a manner identicalto that used to assess BMEC migration. As shown in FIG. 7B, there was aconcentration-dependent decrease in the migration of the BAEC populationin response to the -Jagged oligomer with an approximate 50% reduction inBAEC migration at 5 μM γ-Jaggged oligomer.

When viewed together, these results indicated Jagged-Notch signaling asan anti-migratory event in the endothelium comprising themicrovasculature, but as apro-migratory event in the endothelium oflarge vessels. These experiments demonstrated for the first time thatthere apparently exists a major phenotype difference between small andlarge vessel endothelial cells in response to a ligand-receptorsignaling pathway in the endothelial cell which is modulated during themigratory phase of angiogenesis.

Example 6 Further Characterization of the Disparate Effects Mediated byJagged-induced Signaling in Vitro Using Human Endothelial Cells

To better understand the mechanism utilized by human endothelial cellsto regulate angiogenesis in man, it is important to study the effect ofthe γ-Jaggged oligomer on cell migration using human microvascularendothelial cells and human endothelial cells from large vessels.Although it would be preferable to obtain stable human endothelial cellγ-Jaggged transfectants/transductants using conventional gene transfermethods, none have proven useful with regard to human diploidendothelial cells in vitro. Therefore, the γ-Jaggged oligomer strategyis employed as a means to modify the translational efficiency of thehuman Jagged transcript.

Initially, however, two methods are used to confirm that the γ-Jagggedoligomer is able to reduce the efficiency of Jagged translation. Eachutilizes rabbit anti-Jagged antibodies being prepared against individualsynthetic peptides derived from the extracellular DSL domain, theextracellular cys-poor domain (NH₂-terminal to the transmembrane domain)and the intracellular (i.e., cytoplasmic) domain of the predicted Jaggedprotein sequence. Immunologic methods parallel those previously used forthe production and purification of antibodies against synthetic peptidesderived from sequence analysis of the FGF-1 receptor (Prudovsky et al.,1994, J. Biol. Chem. 269:31720-31724), cortactin (Zhan et al., 1994) andFGF-1 (Imamura et al., 1990, Science 249:1567-1570), and translationproducts are used. Synthetic peptides are prepared as multiple antigenpeptides (MAP) using fmoc MAP resins from Applied Biosystems. Likewise,Notch-1 antibodies are also prepared using sequence from theextracellular LNG domain and intracellular ankyrin repeat domain for MAPsynthesis.

The first method utilizes hybrid selection, using an immobilized Jaggedoligomer to capture the Jagged transcript from HUVEC populations,followed by (³⁵S)-met/cys translation of the Jagged transcript in therabbit reticulocyte system in the absence and presence of varied amountsof the γ-Jaggged oligomer. Immunoprecipitation of the Jagged proteinfollowed by SDS-PAGE autoradiography establishes the ability of theγ-Jaggged oligomer to repress Jagged translation in vitro.

The second method utilizes HUVEC populations metabolically labeled with(³⁵S)-met/cys for Jagged immunoprecipitation from cells exposed tofibrin for 0, 1, 2 and 3 hours. Immunoprecipitation of the Jaggedprotein from the fibrin-induced HUVEC population followed by SDS-PAGEautoradiography permits a comparative assessment of whether pretreatmentof the cells with the γ-Jaggged oligomer represses the level of theJagged protein as a cell-associated polypeptide. The success of thesestrategies is based upon the fact that the Jagged protein sequence isrich in cys residues, and as a result is metabolically labeled to a highspecific activity. Likewise, an accurate molecular weight is assigned tothe Jagged protein since competition with synthetic peptide, pre-immuneserum, as well as denatured γ-Jaggged antiserum, are used as controls todefine the specificity of band assignment. Since the predicted Jaggedtranslation product contains about 1054 amino acids, the molecularweight is in the 135 to 145 kDa range.

The disparate migratory behavior of the BMEC and BAEC populations isconfirmed using stable γ-Jaggged transfectants. Since bovine cells aremore amenable than HUVEC populations to gene transfer methods, thepMEXneo vector (Martin-Zanca et al., 1989, Mol. Cell. Biol. 9:24-33) isused to select for stable BMEC and BAEC γ-Jaggged transfectants aspreviously described (Zhan et al., 1992). Stable clones are obtainedusing G418 resistance to quantify the migratory potential of these cellsrelative to insert-less vector control transfectants. The wound-inducedmigration assay (Example 6; FIG. 7A and 7B) is useful to demonstratethat the serum-induced migration potential of the BMEC γ-Jagggedtransfectants is increased, and the serum-induced migration potential ofthe BAEC γ-Jaggged transfectants is decreased.

The analysis of the effect of the novel protein on human endothelialcells effectively employs the HUVEC population as a model, in comparisonwith HU artery (A) EC and human cells obtained from other anatomicsites, including, e.g., human adipose-derived microvascular endothelialcells (HMEC), human dermis-derived capillary endothelial cells (HCEC)and human saphenous vein (HSVEC) and artery (HSAEC), available fromcommercial and academic sources. The addition of the γ-Jaggged oligomerto these populations of human endothelial cells will be similar to thatdescribed in the protocols involving bovine endothelial cellpopulations. Thus, the ability of they-Jagged oligomer to modulatesprout formation of human capillary, artery and vein endothelial cellsis assessed using the collagen invasion assay described in FIG. 6, andthe migration wound assay described in FIG. 7 supplemented with a Boydenchamber chemotaxis assay as previously described (Terranova et al.,1985, J. Cell Biol. 101:2330-2334). The resulting data, similar to thoseobtained with the bovine endothelial cell populations, confirms theabove-described conclusion (Examples 4 and 5) that reduction in thetranslational efficiency of the Jagged transcript (i) increases humanmicrovascular endothelial cell sprout formation andmigratory/chemotactic potential and (ii) reduces these activities in thehuman endothelial cell populations derived from arteries and veins.

Use of these transfectants permits a more rigorous quantification of thedisparate modulation of migratory potential between small and largevessel endothelial cells using the conventional Boyden chamber assaypreviously used to establish the chemotactic activity of FGF-1(Terranova et al., 1985, J. Cell Biol. 101:2330-2334). In addition, thisapproach also confirms the assessment of the ability of the BAECγ-Jagged and insert-less vector control transfectants to respond to theFGF prototypes as inducers of sprout formation in vitro (FIG. 6).Lastly, this strategy permits an assessment of the migratoryresponsiveness of additional bovine endothelial cells obtained fromalternative anatomic sites, including the portal vein, saphenous arteryand vein, and adipose-derived microvascular endothelial cells. Theability of these cells to induce steady-state levels of Jagged and Notchreceptor transcripts in response to fibrin is also evaluated by RT-PCRanalysis as in Example 3 (FIG. 5).

A nuclear run-on analysis of BMEC and BAEC populations, as well as akinetic analysis of the presence of the Jagged transcript in actinomycinD- and cycloheximide-treated cells in response to fibrin, is employed todetermine whether the induction of the Jagged transcript is due to atranscriptional regulatory event and whether Jagged transcript stabilityis involved in the fibrin response. This analysis is analogous to aprevious study on the post-transcriptional regulation of IL-1α in HUVECpopulations by Garfinkel et al. (1994, Proc. Natl. Acad. Sci. USA91:1559-1563). Nuclear run-on analysis is performed by incubating nucleiobtained from either BMEC or BAEC populations exposed to fibrin for 0,1, 3 and 6 hours with 100 μCi of (⁼P)-UTP for 30 minutes. This isfollowed by the isolation of nascent RNA transcripts, and slot blotanalysis using 5 μg of the linearized, denatured and immobilized JaggedcDNA and hybridization at high stringency with the labeled RNA.Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is used as a positivecontrol, and densitometric values are normalized to the GAPDH signal.Although the level of the Jagged transcript may be difficult to predict,a Jagged signal should be visible. Testing the γ-Jaggged oligomer atvaried levels permits a determination of the ability, if any, of theγ-Jaggged oligomer to access the transcriptional machinery in thissystem.

To determine in those endothelial cell populations that are induced bythe γ-Jagged oligomer to decrease tube formation, it is useful toevaluate whether there is a modification of the steady state transcriptlevels of the immediate-early endothelial differentiation genes (edggenes). This establishes whether the effect of the γ-Jaggged oligomeroccurs during immediate-early or mid-to-late phase of the endothelialcell differentiation pathway and supplements the qualitative data withrespect to the modification of lumen formation in vitro. While the endpoint for this assay will be a qualitative assessment of lumen formationas previously described (Jaye et al., 1985), cells will be harvested asdescribed in Example 3 (FIG. 5) for Northern blot analysis of thepresence or absence of the edi genes, such as the G-protein-coupledorphan receptor, edg-1 (Hla and Maciag, 1990), the transcription factor,edg-2 (Hla et al., 1995, Biochim. Biophys. Acta 1260:227-229),cyclooxygenase-2 (cox-2) (Hla and Neilson, 1992, Proc. Natl. Acad. Sci.USA 89:7384-7388), and tissue collagenase, among others (Hla and Maciag,1990).

Because the data indicate that the γ-Jaggged oligomer acceleratescapillary endothelial cell migration and sprout formation in vitro, theaddition of the Jagged protein to these systems will have the oppositeeffect—inhibiting capillary endothelial cell migration and sproutformation and promoting large vessel-derived endothelial cell migrationin vitro. However, two approaches may be used to evaluate this premise.The first involves the expression and purification of the Jaggedpolypeptide as a recombinant protein, and the second involves theexpression of an extracellular and soluble Jagged construct (asdisclosed in Example 9, infra). Although the predicted Jagged sequencedoes not contain any recognizable post-translational modification motifin the extracellular domain of the protein, such as N-glycosylation, itis possible that a subtle modification of the Jagged protein will affectthe activity of Jagged as a Notch ligand.

Using the recombinant Jagged protein, it is possible to assess itsability to signal through the Notch-1 receptor using a rat myoblastsystem. Since it has been demonstrated that the rat myoblast cell line,C2C12, transfected with the Notch-1 cDNA will not form myotubes whenco-cultured with a lethally irradiated population of murine fibroblasttransfected with the rat Jagged cDNA (Lindsell et al., 1995, Cell80:909-917), it is assumed that the parental C2C12 is aNotch-1-deficient cell line. Therefore, the C2C12 cell represents amodel cell type to assess the biological function of recombinant Jagged.

The C2C12 cell Notch-1 transfectants, but not C2C12 insert-less vectortransfectants, presumably are unable to form myotubes if the recombinantJagged protein is functional as a ligand. Thus, this system also permitsan assessment of the value of Notch-2 as a Jagged receptor.

C2C12 cells are transfected with the full length rat Notch-1 and Notch-2cDNA containing tandem copies of the influenza virus hemagglutinin (HA)epitope and stable transfectants obtained as described (Zhan et al.,1992). The expression of the Notch-1 and Notch-2 receptor transcripts ismonitored by RT-PCR and Northern blot analysis and the protein levelsassessed by immunoprecipitation/Western blot analysis of the HA epitope.The addition of the recombinant Jagged ligand (1 ng to 10 μg titration)permits the Notch-1 and Notch-2 C2C12 cell transfectants to repressmyotube formation, as assessed by morphologic criteria as well as by therepression of the steady-state levels of the myogenic transcript. Thesedata also define the specific activity of the recombinant Jagged proteinfor stability studies (temperature, pH, ionic strength as a function oftime). An appropriate positive control for these experiments is apopulation of lethally-irradiated NIH 3T3 cells transfected with thefull-length Jagged cDNA to the Notch-1 and Notch-2 C2C12 celltransfectants, insuring the attenuation of myotube formation.

After the specific activity of the soluble Jagged protein isestablished, it will be possible to assess the ability of the Jaggedligand in a concentration dependent matter to inhibit microvesselendothelial cell migration, chemotaxis and sprout formation in vitro, asin FIGS. 5 and 6. Effective levels of Jagged protein, similar to thosepreviously functional in the C2C12 cell Notch-1 transfectants, areexpected to also be functional in the human and bovine microvascularendothelial cell systems. A comparable evaluation involves adetermination of the function of the Jagged protein as an inducer oflarge vessel-derived human and bovine endothelial cell migration,chemotaxis, and sprout formation. A concentration-dependent response isindicated. As described above, the co-culture of the large and smallvessel-derived endothelial cells with lethally irradiated NIH 3T3 cellJagged transfectants and insert-less vector transfectants provides asuitable control to demonstrate the disparate role of Jagged-Notchsignaling in the regulation of endothelial cell migration.

Example 7 The Relevance of Jagged-Induced Signaling in Vitro toAngiogenesis In Vivo

Because Jagged was cloned as a fibrin-responsive gene in vitro, an invivo angiogenic system is needed which closely mimics the in vitrosystem. Traditional angiogenesis assays, such as the chickenchorioallantoic membrane (CAM) (Scher et al., 1976, Cell 8:373-382)assay or the rabbit cornea assay (Folkman et al., 1983, Science221:719-725), are useful for an end-point analysis, and are readilyavailable in the art. However, the complexity of the many individualsteps in the angiogenic cascade (FIG. 1), and their control by generegulation, demands a novel in vivo approach that addresses thiscomplexity more specifically.

Plating HUVEC on fibrin has been selected to meet the need for such anin vivo system. It has proven to mimic in vivo, in a reproduciblefashion, the in vitro system we used initially to induce and isolate thehuman Jagged cDNA. The in vivo system involves the subtotal occlusion ofa large vessel, such as a carotid or iliac artery with a thrombus,producing an intimal injury. This is typically followed within two days,by migration of endothelial cells into the three-dimensionalplatelet/fibrin scaffold tube formation. After approximately 4 weeks thesystem characteristically displays tube perfusion, recruitment ofpericytes, and selection of preferred channels with downsizing of minorvessels. Together with the vessels, stromal cells appear as well,contributing to the unique extracellular matrix of this tissue, andmaking this natural, in vivo system (involving revascularization of anexperimental thrombus) ideal for demonstrating the role of Jagged andits receptor(s) in two of the early steps of angiogenesis.

Endothelial migration and tube formation can be separated in time (at 2,4, 6, 8 days after thrombosis), as well as in space. The migrating cellsare primarily located in the central region of the thrombus, whereas theperipheral cells have already formed tubes, as indicated by theappearance of junctions and, almost concomitantly, the arrival ofcirculating red blood cells.

The antibodies developed for use in this experimental system weredesigned for use with known immunoperoxidase or immunofluorescencetechniques to localize endogenous Jagged and Notch (Nabel et al., 1993,Nature 362:844-846). However, an advantage of using this in vivo systemis that the experimentally-induced thrombus can be seeded withgenetically modified cells, γ-Jaggged oligomer, or soluble Jaggedprotein as described above for the in vitro approach, to influence twodistinct phases of the angiogenic cascade in a controlled fashion.

The source of these endothelial cells is from large vessels, but theybehave like capillaries when they migrate and form tubes, until some,but not all, will recruit pericytes and smooth muscle cells and assumethe appearance and function of large vessels again. Clinically, both inthe coronary and in the peripheral circulation, this revascularizationprocess is critical, since successful recanalization of occludingthrombi is highly beneficial to the patient, but its regulation has beenpoorly understood.

Although an expert qualitative pathologic-anatomical evaluation of thevascular morphology is essential in these in vivo experiments, there area number of time points that are amenable to quantitative morphometricanalysis. This is especially relevant since these time points representdistinct stages in this process. At 4, 6, and 8 days, the number ofinvading cells are directly counted using a light microscope to evaluatecross-sections. Using immunohistochemical analysis with the CD34antibody, the.relative number of migrating endothelial cells isquantifiable; and using the leukocyte common antigen, the inflammatorycells can be assessed. Unfortunately, smooth muscle cell α-actin cannotbe used as a reliable marker for myofibroblasts at this stage, sincetheir phenotype is altered. However, by subtraction, the number ofnon-endothelial cells can be determined.

Thus, quantification of this early phase indicates whether, and in whichdirection, the interplay between Jagged and Notch influences themigratory component of the angiogenic process. Using serial sections ofthe same preparations, the proliferative cell nuclear antigen is usefulto evaluate the relative contribution of proliferation to the totalnumber of cells that populate the thrombus. When the thrombus is seededwith transfected cells expressing soluble Jagged, the myc reporter geneis used to recognize and count these components within the system.

Quantification of the functional vascular lumina in a cross-sectionafter 2 and 4 weeks provides additional insight into the relationshipbetween tube formation and the processes of endothelial migration andproliferation during angiogenesis. This comprises a statisticalcomparison of the number of individual lumina, grid point counts, andarea measurements in perfused vessels. Mechanistically, the Jagged/Notchinteraction which initiates tube formation from large vessel endothelialcells in vitro, may prove to be a stop signal for migration andproliferation of the microvasculature.

The endothelial cell site-specific effect of the Jagged-Notch system mayalso be responsible for the control and coordination of themigration/proliferation/tube formation sequence that ultimately leads tothe formation of a new vessel. This can be shown in vivo in arevascularized thrombus murine model system, in which it is possible todeliberately exaggerate or compete with each of the components at themolecular level and at any time point within the process. Indeed, thekinetics of the Jagged/Notch interaction may also be assessable byseeding the thrombus at a later time point with soluble Jaggedtransfectants.

In the mouse, experimental intervention will involve a surgical exposureof previously treated, occluded carotid artery for an injection of asmall volume of either lethally irradiated transfectants, recombinantprotein or γ-Jaggged oligomer into the site. However, the occludedvessel cannot bleed due to incomplete revascularization. Appropriatecontrols for the repetitive minor surgical trauma are possible in thesame mammal on the contralateral carotid, using cells transfected withan inactive, but minimally altered mutant, inactive recombinant protein,or sense or inactive mutant γ-Jaggged oligomers respectively.

While the model is useful to examine the formation of a newthree-dimensional network of functioning vascular tubes, an additionalmodel for the re-endothelialization of the intima of a large vessel isneeded, since Jagged/Notch appears to regulate this process in theopposite direction. Since murine vessels are too small for precise,selective de-endothelialization, the gently ballooned rat thoracic aorta(access from the carotid with a French 2 Edwards balloon) is anappropriate test system since it offers unequivocal starting points, andreasonably accurate quantification (see Schwartz et al., 1978, Lab.Invest. 38:568-580).

To assess the ability of the Jagged ligand to modify the migration ofendothelial cells, thus influencing their ability to form a capillarynetwork and/or to cover a de-endothelialized surface, one of severalmethods is indicated. In a first method, a therapeutically-effectiveamount of soluble Jagged ligand is administered intravenously to miceand/or rats prior to and/or following thrombosis or balloon injury. Inan alternative method, a thrombotic occlusion in a mouse is seeded withan effective amount of lethally irradiated NIH 3T3 cell solubleJagged:myc transfectants. While in a third method, in both rats andmice, a distal site is seeded with an effective amount of lethallyirradiated NIH 3T3 cell soluble Jagged:myc transfectants onto asubcutaneous fibrin matrix implant, which has been pretreated withlethally irradiated NIH 3T3 cells transfected with a hst-sp-FGF-1construct using the nude mouse (Forough et al., 1993, J. Biol. Chem.268:2960-2968).

It is known that the NIH 3T3 cells hst-sp-FGF-1 transfectants (10⁵cells) are able to secrete FGF-1 as an extracellular angiogenesissignal, and establish within 5 to 10 days an aggressive capillarynetwork (Forough et al., 1993). This is a result of the ligation of thesignal peptide (sp) sequence from the hst/KS5 (FGF-4) gene to FGF-1,which directs the traffic of the hst-sp-FGF-1 chimera into the ER-Golgiapparatus for proteolytic processing of the hst/KS5-sp-sequence andrelease of FGF-1 as a soluble, extracellular protein. The efficacy ofthis construct has been established in vivo (Nabel et al., 1993;Robinson et al., 1995, Development 121:505-514).

In the third method, following thrombotic occlusion, the NIH 3T3 cellsoluble Jagged:myc transfectants (10⁶-10⁷ cells) are injected into theangiogenic site, enabling the cells to express and secrete the solubleJagged protein into the vasculature. The levels of plasma-derived Jagged(tail vein samples) are monitored by ELISA using the myc-epitope andJagged antibodies. The rats are then assessed over time (e.g., 1 to 10days at 2 day intervals) for re-endothelialization of the denuded arteryusing Evan's blue staining. The degree of angiogenesis in the occlusionzone in the murine vessels is assessed using morphometric analysis ofindividual endothelial cells and of the fully developed capillaryvessels in histological sections. Indeed, analysis by transmissionelectron microscopy will clearly demonstrate the involvement ofendothelial cell migration and sprout formation in this system.

The assessment of the pharmacologic administration of intravenoussoluble Jagged in the first method is based upon a similar end point,but utilizes a sufficient amount of recombinant Jagged to saturate boththe Notch-1 and Notch-2 receptor Jagged-binding sites. The number andaffinity of Jagged-binding sites on the surface of the murineendothelial cell are quantified in vitro by Scatchard analysis of murineaorta-derived endothelial cells and adipose-derived microvascularendothelial cells using competitive (¹²⁵I)-Jagged binding by the methoddescribed for FGF-1 (Schreiber et al., 1985, Proc. Natl. Acad. Sci. USA82:6138-6142).

The apparent lack of regulation of the Notch-1 and Notch-2 transcriptsin the HUVEC population (FIG. 5), predicts a high affinity Kd (pM) withapproximately 5-20,000 Notch-binding sites per cell. The radiolabellingof the Jagged polypeptide utilizes the lactoperoxidase method, followedby removal of free (¹²⁵I) by Sephadex G-50 gel exclusion chromatography.This provides a pharmacologic range for the administration of theligand. In addition, the availability of (¹²⁵I)-Jagged will demonstratethe expected pharmacokinetics of intravenous Jagged using methodspreviously successful for FGF-1 (Rosengart et al., 1989, Circ. Res.64:227-234).

In sum, these models should provide an in vivo correlate and in vivomodels for Jagged function, demonstrating a predicted increase (25%-35%)in lumen re-endothelialization, and a similar decrease in the formationof capillary structures. In comparisons between the in vivorevascularization and re-endothelialization experiments in normotensiveanimals, and in their spontaneously hypertensive rat counterparts (SHR,commercially available from Charles River with guaranteed hypertension),it has been shown that hypertension has a direct, albeit subtle, effecton the aortic endothelium of these model animals (Haudenschild et al.,1981, Hypertension 3:148-153). The aortic re-endothelializationexperiments can be repeated in these rats without modification and withhypertension as the only added variable, however, the thrombusrevascularization experiments must also be performed in these rats,since there is no comparable murine hypertension model available. Thethrombi have been shown to be readily reproducible in mice, rats andrabbits. Thus, species differences do not pose a technical problem inthe in vivo model systems.

Example 8 Expression of Soluble Jagged in the NIH 3T3 Cell Line

To determine the effects of a secreted, extracellular form of Jagged, amodified form of the nucleic acid encoding Jagged was synthesized,transfected into the NIH 3T3 cell line, and then cells were selectedthat produced the protein. To track and monitor the fate of this Jaggedmolecule, a myc tag (reviewed by Kolodziej and Young, 1991, Meth.Enzymol. 194:508-519) was also introduced at the 3′ end of the gene. Inorder to do this, several modifications of the jagged gene werenecessary, these are: (1) a Kozak sequence (Kozak, 1989, J. Cell Biol.108:229-241) was engineered onto the 5′ end of the gene to ensureefficient transcription, (2) a myc epitope tag was placed at the 3′ end,and (3) cloning sites were engineered on both the 5′ end (EcoRI, BamHI,SalI sites) and the 3′ end (XhoI site).

The primer pairs used for this construction were as follows.

The primers used to construct the 5′ end of the molecule were: forwardprimer 5′-GACTATGCGAATTCGGATCCGTCGACGCCACCATGG-3′ (SEQ ID NO:13), andreverse primer: 5′-CAAGTTCCCCCGTTGAGACA-3′ (SEQ ID NO:14).

The primers used for construction of the 3′ end of the molecule encodingJagged-myc tag were as follows: reverse primer5′-GCATAGTCCTCGAGTTACAAGTCTTCTTCAGAAATAAGCTTTTGTTCTACGA TGTACTCCATTCG(SEQ ID NO:15), and forward primer 5′-ATGGACAAACACCAGCAGAA (SEQ IDNO:16). PCR cycling reactions were performed as described previouslyelsewhere herein.

The 5′ reaction amplification product was digested with EcoRi and BglII.The 3′ amplification product was digested with XhoI and AccI restrictionendonucleases. The two amplicons were ligated into a similarly digestedJagged template construct using a standard protocol well-known in theart. The final gene product was then digested with EcoRI and XhoI andthen ligated into the eukaryotic expression vector pMexNeo2. Thisexpression construct was then transfected into the NIH 3T3 cell line andcells were grown in selection media containing G418 as describedelsewhere herein and/or per standard protocols well-known in the artsuch as those described in Sambrook et al. (1989, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York), and Ausubelet al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons,New York).

Calcium mediated DNA transfer into NIH 3T3 cells was followed by growthin selective media and resulted in selection of clone MW38-1.1 (SEQ IDNO:17). The data disclosed herein demonstrate that clone MW38-1.1synthesized a protein having the anticipated characteristics (e.g.,molecular weight, amino acid sequence [SEQ ID NO:18], and the like) ofJagged-myc tag and that the protein was released into the surroundingmedium, termed “conditioned media.”

The data disclosed herein demonstrate that transfectant cells expressingclone MW38-1.1 exhibited a unique phenotype. The transfectants grosslyformed chord-like structures in vitro correlating with the presence ofpseudo-lumens by ultrastructure analysis (see Example 9, FIGS. 10B and10D). In addition, the cells were able to induce wild type NIH cells topartially assume this phenotype. Therefore, the data disclosed hereindemonstrate that MW38-1.1 transfectant cells are an outstanding resourceboth for the production and isolation of the soluble Jagged (alsoreferred to as “sol-jag”) protein (SEQ ID NO:18), and for their abilityto modulate the differentiation pattern of adjacent cells.

Example 9 In vivo and in vitro Effects of Soluble Jagged Expression

The experiments presented in this example may be summarized as follows.

As discussed previously elsewhere herein, Jagged-Notch interactionsregulate a transmembrane ligand-receptor signaling pathway involved inthe regulation of cell fate determination as well as myoblast andendothelial cell differentiation. To further examine the role of thetransmembrane ligand, Jagged-1, in the regulation of endothelial celldifferentiation (Zimrin, et al., 1996, J. Biol. Chem. 271:32499-32505),NIH 3T3 cells were stably transfected using a nucleic acid encoding atruncated form of Jagged-1 (FIGS. 13B and 13C, [SEQ ID NO:17]), whichresults in the secretion of a soluble form of the protein, i.e., solubleJagged (FIG. 13A, [SEQ ID NO:18]). Comparison of gene expression byserial analysis demonstrated that pro-α-2(I) collagen was repressed insoluble Jagged-1 transfectants. The data disclosed herein furtherdemonstrate that when plated on extracellular matrices, soluble Jagged-1transfectants formed prominent chord-like structures of Type I collagenbut did not form such structures when plated on fibrin, fibronectin orvitronectin.

While the soluble Jagged-1 transfectants exhibited growth kineticssimilar to control cells and were unable to grow in soft agar, the cellswere less sensitive to contact inhibition of growth in vitro and solubleJagged-1 allografts formed tissue masses in nude mice after a prolongedlatency period. Because these tumor-like structures exhibited anabundance of host-derived microvascular endothelial cells, theangiogenic potential of the soluble Jagged-1 transfectants was assessedby implantation of lethally-irradiated transfectants in the chickchorioallantoic membrane assay. These irradiated transfectant cells werenot only able to induce angiogenesis but were also able to direct theformation of large macrovessel-like structures.

These data disclosed herein indicate that Jagged-1 can initiateangiogenesis by the organization of matrix-sensitive cell-cellinteractions including its ability to promote the development ofchord-like structures.

The Materials and Methods used in the experiments presented herein arenow described.

Soluble Jagged-1 Plasmid Construction

The soluble myc epitope-tagged Jagged expression vector was generatedusing two separate sequential polymerase chain reaction (PCR) protocols.Overhang PCR was used to place a consensus Kozak sequence (Kozak, 1989,J. Cell Biol. 108:229-241) 5′ to the Jagged-1 open-reading frame (ORF),and to truncate Jagged-1 immediately 5′ to the transmembrane domain.This construct was assembled by ligating the PCR-modified 5′ and 3′amplicon into the shuttle plasmid, MW27, which consists of thefull-length Jagged-1 cDNA in pBlue Script and was subcloned into theeukaryotic expression vector pMexNeo2 (Martin-Zanca et al., 1989, Mol.Cell. Biol. 9:24-33) using the newly engineered 5′ EcoRI and 3′ XhoIsites to produce the final product. The forward primer used for the 5′modifications was 5′-GACTATGCGAATTCGGATCCGTCGACGCCACCATGGGTTCCCCACGGACACGCG-3′ (SEQ ID NO:19) and reverse primer was 5′-CAAGTTCCCCCGTTGAGACA-3′(SEQ ID NO:20), where the Kozak sequence is underlined. The forwardprimer used for the 3′ modification was 5′-ATGGACAAACACCAGCAGAA-3′ (SEQID NO:21) and reverse primer was5′-TAGTGCTCGAGCTATTACAAGTCTTCTTCAGAAATAAGCTTTTGTTCATCTG TTCTGTTCTTCAG-3′(SEQ ID NO:22), where the myc epitope is underlined. The template usedfor PCR was the complete human Jagged-1 ORF originally obtained form Dr.G. Gray, Yale University.

PCR reactions were performed using Vent polymerase (New England Biolabs,Beverly, Mass.) in 1×vent buffer as recommended by the manufacturer. PCRthermal cycling parameters consisted of 94° C. (1 minute) followed by 35cycles at 94° C. (30 seconds), 62° C. (30 seconds), 72° C. (30 seconds)followed by a 10 minute hold at 72° C. before termination at 4° C.

The 5′ PCR-modified product was digested with EcoRI and BglIII,electrophoretically resolved on a 1% (w/v) agarose gel, electroeluted,and then ligated with a similarly digested MW27 to create MW13 usingstandard protocols (Sambrook et al., 1989, Molecular Cloning, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The modified3′ PCR-amplified product was processed similarly except that therestriction digestion step utilized XhoI and AccI. The ligation wasperformed with a similarly digested MW13 to yield MW32. This5′-Kozak-truncated Jagged-1 3′-myc-tagged pBlueScript construct wasdigested with EcoRI and XhoI and ligated into pMexNeo. All restrictionenzymes and buffers were obtained from New England Biolabs and twosoluble Jagged-1 transfectant clones, 38-1 and 38-4, and one insert-lessvector transfectant clone were used for experimentation.

Cell Transfection, Immunoprecipitation, Immunoblot Analysis, and MatrixPreparation

NIH 3T3 cells were transfected with clone 38 (also referred to as“MW38”) using a calcium-phosphate kit (Stratagene, La Jolla, Calif.) andresulting transfectants were selected using G418 (Gibco/BRL,Gaithersburg, Md.) selection. Stable soluble Jagged-1 transfectants weregrown and maintained in DMEM (GIBCO/BRL, Gaithersburg, Md.) supplementedwith 400 μg/ml G418 and 10% (v/v) fetal bovine serum (FBS) (HyClone,Logan, Utah). G418 resistant cells were grown to confluency in DMEMcontaining 10% (v/v) FBS, the cells were washed twice inphosphate-buffered saline (PBS), and the cells were then incubated with[35S] labeling media consisting of cys- and met-free DMEM supplementedwith 1×Nutriderma (Gibco/BRL, Gaithersburg, Md.) and 0.4 μCi/ml of[³⁵S]-met/cys mixture (DuPont-New England Nuclear). After 4 hours, thelabeling medium was removed, the cells were washed once with ice coldPBS, and the cells were scraped into 1.0 ml of PBS. The cells werepelleted and the cell pellets were resuspended in RIPA lysis buffercontaining 1 mM PMSF, 10 μg/ml aprotinin and 1 μg/ml leupeptin (SigmaChemical Co., St. Louis, Mo.). The samples were clarified bycentrifugation (13,000×g for 10 minutes) and then incubated with 30 μlof Protein-A Sepharose (Pharmacia LKB Biotechnology, Inc., Piscataway,N.J.) which had been complexed with 9E10 anti-myc monoclonal antibodies(Oncogene, Boston, Mass.). The immunoprecipitates were washed four timeswith RIPA buffer, and the immunoprecipitates were then dissolved in 50μl of 2×SDS sample buffer. The eluted proteins were resolved in 8%SDS-PAGE as described previously (Laemmli, 1970, Nature 227:680-685).

To assess the secretion of soluble Jagged-1, the conditioned medium (1ml) from the [³⁵S]-met/cys-labeled cells was collected in 1 mM PMSF and10 μg/ml aprotinin then incubated with 50 μl of Protein-A Sepharose andtreated as outlined above for cell lysates except that theimmunoprecipitates were washed six times prior to being dissolved in SDSsample buffer.

Confluent monolayers of soluble Jagged-1 NIH 3T3 and insert-less vectorNIH 3T3 cell transfectants were lysed by scraping in 1 ml of SDS-PAGEsample buffer containing 2% (v/v) mercaptoethanol and the samples wereboiled for 10 minutes. To equalize for protein load, cells wereindependently lysed by scraping into 20 mM Tris buffer, pH 7.5,containing 1% (v/v) Triton X100, the protein concentration was measuredusing the Coomassie Protein Assay Kit per the manufacturer'sinstructions (Pierce Chemical Co., Rockford, Ill.), equal protein loadswere resolved using 6% acrylamide (w/v) SDS-PAGE. The proteins weretransferred to Hybond C membranes (Amersham, Arlington Heights, Ill.)using standard methods. The blots were immunostained using the SP 1.D8mouse monoclonal antibody specific for pro-α-1(I) collagenamino-terminal extension peptide (Developmental Studies Hybridoma Bank,University of Iowa). Pro-α-1(I) collagen was visualized using ahorseradish peroxidase-conjugated goat anti-mouse IgG (Bio-RadLaboratories, Richmond, Calif.) and an enhanced chemiluminescence (ECL)detection system (Amersham).

Cell culture dishes were coated with 10 μg/cm² of human fibronectin for2 hours, the fibronectin was removed, and the plates were washed threetimes with sterile PBS. Collagen gels were formed in 6-well plates bymixing 8 volumes of type I collagen (Vitrogen 100, Collagen Corporation,Palo Alto, Calif.) with 1 volume of 10×DMEM (Gibco/BRL) and 1 volume ofsodium bicarbonate (11.8 mg/ml) on ice and then quickly dispensing (1.5ml) of the mixture into each well of the individual cell culture dishes.The collagen mixture was allowed to gel for 1 hour prior to use. Softagar growth assays were performed as described previously (Forough etal., 1993, J. Biol. Chem. 268:2960-2968).

Serial Analysis of Gene Expression (SAGE)

The SAGE method was performed as previously described (Zimrin andMaciag, 1996, J. Clin. Invest. 97:1359). Briefly, polyA⁺ RNA derivedfrom insert-less vector control and from soluble Jagged-1 NIH 3T3 celltransfectants converted to double stranded (ds)-cDNA (cDNA SynthesisSystem, BRL) was purified by reversed phase HPLC using 5′-biotin-dT₁₈(Integrated DNA Technologies, Inc., Coralville, Iowa). The cDNA wascleaved with N1aIII, and the 3′-biotinylated fragments were captured onstreptavidin-coated magnetic beads (Dynal, Oslo, Norway). The bound cDNAwas divided into two pools, and one of the following linkers containingrecognition sites for BsmFI and a N1aIII complementary terminus wasligated to each pool: linker 1,5′-TTTGGATTTGCTGGTGCAGTACAACTAGGCTTAATAGGGACATG-3′ (SEQ ID NO:23), 5′TCCCTATTAAGCCTAGTTGT ACTGCACCAGCAAATCC (amino-C7)-3′ (SEQ ID NO:24) andlinker 2, 5′-TTTCTGCTCGAATTCAAGCTTCTAACGATGTACGGGGACATG-3′ (SEQ IDNO:25), 5′ TCCCCGTACATCGTTAGAAGCTTGAATTCGA GCAG (amino-C7)-3′ (SEQ IDNO:26).

SAGE tags were released with BsmFI, the tag overhangs were filled inusing T7 polymerase, and the tags were ligated using T4 DNA ligase (BRL)overnight at 25° C. The SAGE tags were diluted and amplified by PCR for28 cycles (primers: 5′-GGATTTGCTGGTGCAGTACAACT-3′ [SEQ ID NO:27] and5′-CTGCTCGAATTCAAGCTTCTAAC-3′ [SEQ ID NO:28]). The product wasfractionated using polyacrylamide gel electrophoresis (PAGE), and the104 bp product containing two tags ligated tail to tail (ditag) wasexcised and extracted from the gel. The product was cleaved with N1aIII,and the ditags were purified by gel electrophoresis, excised from thegel, and then self-ligated to produce ditag concatamers (Velculescu etal., 1995, Science 270:484-487; Velculescu, 1997, Cell 88:243-251). Theconcatenated products were separated by PAGE, and products ranging fromabout 300 bp to about 800 bp were excised from the gel and cloned intothe SphI site of pZero (Invitrogen, Carlsbad, Calif.).

Colonies were screened for insert size by PCR using M13 forward and M13reverse primers. Clones were introduced into 25 μl PCR reactionscontaining 0.5 μM M13 forward and reverse primers and the samples werethen subjected to thermal cycling (25 cycles) consisting of 20 secondsat 95° C., 1 minute at 52° C. and 1 minute at 72° C. Clones selected onthe basis of insert size were subjected to automated fluorescent DNAsequence analysis using rhodamine dideoxynucleotide terminator chemistryaccording to the instruction of the manufacturer (Applied Biosystems,Inc., Foster City, Calif.).

The sequence files were analyzed by means of the SAGE program group,which identifies the anchoring enzyme site with the proper spacing,extracts the two intervening tags, and records them in a database. Thepotential identities of the tags was established by their presence inGenBank or DbEST databses (release 109).

Assessment of Soluble Jagged-1 NIH 3T3 Cell Transfectant Behavior inVivo

The soluble Jagged-1 NIH 3T3 cell transfectants were grown to confluenceunder G418 selection and, 24 hours prior to injection, the medium waschanged to DMEM containing 10% (v/v) FBS. The transfectants were washedwith PBS, harvested by trypsin digestion, and then resuspended insterile/pyrogen-free PBS prior to injection. The cells were greater thanabout 95% viable as determined by Trypan Blue exclusion and were free ofmycoplasma and indigenous murine viruses including mouse hepatitis,adenovirus, pneumonia, cytomegatovirus and Sendai (Anmed/Biosafe Inc.,Rockville, Md.).

Female athymic nude mice (nu/nu) between 8-12 weeks of age (NCI-FCRDC)received 150 mg/kg of cyclophosphamide in pyrogen-free water by theintraperitoneal route 24 hours prior to injection. Injection of a 200 μlcell suspension (10⁶) was administered intradermally into the rightflank.

Following euthanasia, tissue growths were exposed by dissecting alongthe subcutaneous tissue plane and the tissue masses were removed, fixedin 10% (v/v) buffered formalin, and the tissue was processed forparaffin sectioning and hematoxylin and eosin staining. Representativeportions of these masses were also embedded in O.C.T. compound (MilesScientific, Elkhart, Ind.) and snap frozen in 2-methylbutane (E.M.Science, Gibbstown, N.J.) on dry ice. Frozen sections were placed ontoglass slides, fixed in chilled acetone, and dried.

Immunohistochemistry was performed using the ABC system (VectorLaboratories, Burlingame, Calif.) and a 1:200 dilution of an antibody(PharMingen, San Diego, Calif.) to rat-derived endothelial cell-specificmarker CD31/PECAM.

The chick chorioallantoic membrane (CAM) angiogenesis assay wasperformed as described previously (Brooks et al., 1994, Science264:569-571; Jadhav et al., 1999, FASEB J. 13:4) and utilized 2.5×106lethally irradiated soluble Jagged-1 NIH 3T3 cell transfectants per CAM.Recombinant human FGF-2 and insert-less vector NIH 3T3 celltransfectants served as positive and negative controls, respectively.The assay was harvested 4 days post-implantation and the angiogenicindex was quantitated by computer-assisted morphometric analysis ofvessel number.

The Results of the Experiments presented herein are now described.

Angiogenesis is an integral part of physiologic and pathologic processessuch as embryonic development, wound repair, solid tumor growth andchronic inflammation and involves the ability of the endothelial cell tocoordinate migration, proliferation, and differentiation pathways toform new vascular structures (Zimrin and Maciag, 1996, J. Clin. Invest.97:1359; Folkman and D'Amore, 1996, Cell 87:1153-1155). While theability of the angiogenic growth factors, vascular endothelial growthfactor (VEGF) and fibroblast growth factor (FGF) to initiate endothelialcell migration and growth are well described (Maciag et al., 1979, Proc.Natl. Acad. Sci. USA 76:5674-5678; Chen and Chen, 1987, Exp. Cell Res.169:287-295), the identification of factors involved in the regulationof the tubular, chord-like vascular phenotype has been difficult toaccess. Data disclosed elsewhere herein demonstrate that thetransmembrane protein, Jagged-1, a ligand for its transmembrane receptorNotch (Lindsell et al., 1995, Cell 80:909-917), is involved in theregulation of human endothelial cell differentiation in vitro (see alsoZimrin and Maciag, 1996, J. Clin. Invest. 97:1359). Jagged-Notch is anevolutionarily conserved intercellular signaling pathway responsible forthe regulation of developmental cell fate decisions in vivo (Weinmaster,1998, Current Opinion in Genetics & Development 8:436-442) and cellulardifferentiation in vitro (Carlesso et al., 1999, Blood 93:838-848;Milner et al., 1996, Proc. Natl. Acad. Sci. USA 93:13014-13019).

During the cloning of the human Jagged-1 gene, two cDNA clones wereisolated which contained identical deletions resulting in the insertionof 15 novel amino acids followed by a premature termination of theJagged-1 sequence prior to the domain encoding the transmembrane andintracellular sequences (Example 2; see also Zimrin and Maciag, 1996, J.Clin. Invest. 97:1359). Since this truncated Jagged-1 cDNA contained theJagged-1 signal peptide sequence, cells transfected with this constructwere prepared such that the cells would secrete the truncated ectodomainof Jagged-1 as a soluble and extracellular form of the Jagged-1 proteinthereby eliminating the transmembrane constraints imposed upon thenon-truncated Jagged-1 ligand to signal by an intercellular pathway. Thedata disclosed herein demonstrate that human soluble Jagged-1 is anangiogenesis factor in vivo which is able to influence the formation ofa chord-like phenotype in vitro.

SAGE Analysis of Soluble Jagged-1 NIH 3T3 Transfectants

The soluble Jagged-1 transfectants were analyzed for Jagged-1 expressionby immunoprecipitation of [³⁵S]-cys/met-labeled cells. As shown in FIG.9, SDS-PAGE analysis of the myc epitope immunoprecipitants resolved aband of approximately 130 kDA in both cell lysate and conditioned mediumwhich band corresponds to the size predicted by the mass of the solubleJagged-1 myc epitope translation product.

Analysis of differential gene expression by SAGE also demonstrated thatthe soluble Jagged-1 transfectants were able to differentially express227 transcripts of comprising either known or novel sequences. Theseresults were posted at the web site for the Maine Medical CenterResearch Institute, and a selected number are listed in Table 1.

TABLE 1 Most Frequently Observed SAGE Tags RNA Source Tags SequencedDiscrete Tags mRNA Species Insert-less Vector 1428  982 197 SolubleJagged-1 3150 1647 336 Totals 4578 2629 533 SEQ Tag Count ID NO: Acc.No. Description Tags Predominant in Soluble Jagged-1 NIH 3T3 CellTransfectants TGGATCAGTC 14  34 M62952 Mus musculus ribosomal proteinL19 TAAAGAGGCC 9 35 U67770 Mus musculus ribosomal protein S26 (RPS26)mRNA CCTGATCTTT 8 36 X06406 Mouse mRNA for translational controlled 40kDa protein TGTAACAGGA 8 37 X04648 Mouse mRNA for IgG1/IgG2b Fc receptor(FcR) TCTGTGCACC 6 38 U93864 Mus musculus ribosomal protein S11 mRNACCAAATAAAA 6 39 U13687 Mus musculus DBA/2J lactate dehydrogenase-ACTAATAAAAG 6 40 X54691 Mouse COX4 mRNA for cytochrome c oxidase subunitGCCAAGGGTC 6 41 L08651 Mus musculus large ribosomal subunit protein mRNAGTCTGCTGAT 5 42 X75313 M. musculus (C57BL/6) GB-like mRNA AAGGAAGAGA 443 X51438 Mouse mRNA for vimentin TGAAATAAAC 4 44 M33212 Mouse nucleolarprotein N038 mRNA CACCACCACA 4 45 X05021 Murine mRNA with homology toyeast L29 ribosomal prot. CCTCAGCCTG 4 46 X52886 Mus musculus mRNA forcathepsin D. CTCTGACTTA 4 47 Y16256 Mus musculus mRNA for basiginGTGGGCGTGT 4 48 M33330 Mouse insuloma (rig)mRNA TCCTTGGGGG 4 49 U60001Mus musculus protein kinase C inhibitor (mPKCI) mRNA Tags Predominant inControl Insert-less Vector NIH 3T3 Cell Tranfectants CGCCTGCTAG 3 50X58251 Mouse COL1A2 mRNA for pro-alpha-2(I) collagen AAAAAAAAAA 2 51AF0253 Mus musculus tssk-1 and tssk-2 kinase substrate mRNA AAGCAGAAGG 252 M16465 Mouse calpactin I light chain (p11) mRNA complete CAGGACTCCG 253 M26270 Mouse stearoyl-CoA desaturase (SCD2) mRNA GAAGCAGGAC 2 54D00472 Mouse mRNA for cofilin GGATATGTGG 2 55 M20157 Mouse Egr-1 mRNAGTTCTGATTG 2 56 U88588 Mus musculus cdr2 mRNA

Note. Tags correspond to the 10 base pairs of DNA sequence dataimmediately following the NlaIl cleavage site. The count refers to thenumber of instances the tag appears in the SAGE database. Accessionnumbers (Acc. No.) are the GenBank designations referring to the mRNAidentified in the description column. SAGE was conducted using cDNAderived from NIH3T3 cells that had been stably transfected with thepMexNeo insert-less parent vector or the sJ-1 construct. A total of 4578Tags were sequenced consisting of 1428 from pMexneo and 3150 pMexNeosJ-1 transfected cell derived cDNA. Analysis of the data revealed atotal of 2629 discrete tags comprised of 982 separate mRNA species frompMexNeo and 1647 separate mRNA species from the sJ-1-transfectedcell-derived cDNA. Linkage to GenBank database version 109 yielded atotal of 533 matches with documented mouse mRNA species composed of 197mRNA species from the pMexNeo-derived tags and 336 mRNA species from thesJ-1-derived tags. A p-value of 0.05 or less was chosen as the cutofffor statistically relevant alterations and only the most predominanttags are shown.

The 163 known transcripts expressed at an enhanced level by the solubleJagged-1 NIH 3T3 transfectants in the SAGE analysis include, but are notlimited to, cathepsin D (Acc. No. Z53337), and vimentin (Acc. No.X51438).

Moreover, the 64 known transcripts with apparent reduced levels ofexpression in the soluble Jagged-1 NIH 3T3 transfectants include, butare not limited to, pro-α-2(I) collagen (Acc. No. X58251).

Because SAGE analysis can provide insight into the presence of knownmetabolic or signaling pathways, it is important to note that the datadisclosed herein demonstrate that the following transcripts: sps1/ste20related kinase, YSK2 (Acc. No. U49949), enhancer of split-Groucho, ESG(Acc. No. X73360), Mus musculus protein kinase C inhibitor, mPKCI (Acc.No. 6001), type IV collagenase (Acc. No. X83424), and connexin 32 (Acc.No. M63802), were present in the soluble-Jagged-1 NIH 3T3 transfectantswhereas the fibroblast growth factor receptor 1, FGFR-1 (Acc. No.M33760) and the IκB-β (Acc. No. U 19799) transcripts were not present inthe transfectants.

Since pro-α-2(I) collagen expression appeared to be prominent among therepressed transcripts, the expression of the translation product wasexamined further using insert-less vector and soluble-Jagged-1 NIH 3T3transfectants. Because antibodies specific for pro-α-2(I) collagen arenot available, the expression of the type I collagen translation productwas assessed using immunoblotting to detect the pro-α-1(I)amino-terminal extension peptide using the SP 1.D8 monoclonal antibodyspecific for the extension peptide as described elsewhere herein. Thedata disclosed herein demonstrate that immunoblot analysis detectedexpression of pro-α-1(I) collagen translation product in insert-lessvector NIH 3T3 transfectant cells (FIG. 9, lane 1), but the expressionof this type I collagen was not detected in soluble Jagged-1 NIH 3T3transfectant cells transfected with either soluble Jagged-1 clone 38-1(FIG. 9, lane 2) or clone 38-4 (FIG. 9, lane 3).

NIH 3T3 cell Soluble Jagged-1 Transfectants Exhibit the Formation of aMatrix-dependent Chord-like Phenotype

Since pro-α-2 (I) collagen expression appeared to be prominent among therepressed transcripts and since collagen matrices are known modifiers ofcellular phenotype in vitro (Michalopoulos and Pitot, 1975, Exp. CellRes. 94:70-78), the soluble Jagged-1 transfectant cells were plated ontype I collagen. As shown in FIG. 10D, the soluble Jagged-1transfectants plated on collagen exhibited a chord-like phenotype withthe formation of an interlacing arborizing pattern. This chord-likephenotype was also observed when the soluble Jagged-1 transfectants wereplated on plastic at low seed density (FIG. 10B) in which groups ofcells organize into chord-like arrays one to two cells in width. Whilethese structures progressed through the arboring phase, the monolayerassumed a normal NIH 3T3 cell phenotype as the population density nearedconfluence. On occasion, these structures were readily visible in theconfluent monolayer and extended several millimeters in length. Incontrast, soluble Jagged-1 transfectants did not exhibit a chord-likephenotype on either fibrin, fibronectin or vitronectin-coated surfaces.Likewise, neither wild type NIH 3T3 cells nor insert-less vector NIH 3T3cell transfectants exhibited this chord-like phenotype either on plastic(FIG. 10A) or on a collagen type-1 matrix (FIG. 10C).

NIH 3T3 Cell Soluble Jagged-1 Transfectants Modify Angiogenesis

A comparative assessment of the proliferative potential of the solubleJagged-1 transfectants with insert-less vector transfectants revealedthat the population doubling time was not altered when cells weresubconfluent and this was consistent with the absence of a transformedin vitro phenotype including the failure of the Jagged-1 transfectantsto grow in soft agar. However, the soluble Jagged-1 transfectants werenot sensitive to contact inhibition of growth (FIG. 11). The datadisclosed herein demonstrate that the soluble Jagged-1 NIH 3T3 celltransfectants exhibited the ability to grow to significantly higher celldensities than the control insert-less vector NIH 3T3 celltransfectants.

Because of this difference in growth kinetics, the potential of solubleJagged-1 NIH 3T3 cell transfectants to form tumors in nude athymic micewas assessed. Data disclosed herein demonstrate that in transplantationstudies using nude mice, the soluble Jagged-1 transfectants were able toform tissue masses (FIG. 12A) but only after an extended latency periodof approximately 8 weeks. Full necropsy of these animals did not revealany evidence of local or distant metastases and gross dissection ofthese tissue masses revealed prominent angiogenesis characterized by 1-2large feeder vessels, each giving rise to a rich percolating network ofsmaller vessels visible on the surface of the tissue mass (FIG. 12A).Histologic examination further revealed large numbers of capillaries onthe surface that penetrated into the body of the tissue (FIG. 12B).Immunohistochemical analysis of the endothelial cell-specific marker,CD31 (PECAM), demonstrated not only the presence of microvessels butalso a plethora of CD31-positive cells organized as a collection ofeither noncontiguous single cells or sharply angulated short lineararrays (FIGS. 12C and 12D). Interestingly, unlike the well-formedintratissue mass microvessels, very few of these groups of CD31-positivecells contained blood, nor were they associated with intratissue massblood spaces (FIGS. 12C and 12D).

Since primary in vitro cell isolates of the soluble Jagged-1transfectants obtained from these tissue masses by G418 selectiondemonstrated their ability to form chord-like structures andre-implantation into nude mice demonstrated their ability to developangiogenic tissue masses with a similar latency period, the angiogenicpotential of the soluble Jagged-1 transfectants was determined using theconventional chorioallantoic membrane (CAM) angiogenesis assay (Brookset al., 1994, Science 264:569-571; Jadhav et al., 1999, FASEB J. 13:4),which is an art-recognized model of angiogenesis. Implantation oflethally irradiated soluble Jagged-1 transfectants yielded a prominentangiogenic response similar to the positive control, fibroblast growthfactor 2 (FGF-2), while the insert-less vector transfectants did not.Unexpectedly, the soluble Jagged-1 CAM also exhibited the formation ofprominent macrovessels, a novel and unusual feature which has not beenpreviously observed with other angiogenic factors such as fibroblastgrowth factor (FGF) and vascular endothelial growth factor (VEGF) (Oh etal., 1997, Dev. Biol. 188:96-109).

Although the human Jagged-1 transcript as a gene is modified during theearly stage of in vitro angiogenesis (Zimrin et al., 1995, Biochem.Biophys. Res. Commun. 213:630-638), the data disclosed herein establisha role for the soluble form of Jagged-1 as a modifier of chordformation. Interestingly, SAGE analysis disclosed herein suggestsalterations in gene expression that may be relevant to the function ofJagged-1 during cell differentiation in vitro. In addition to therepression of type I collagen gene expression, the steady state levelsof the transcripts for FGFR-1 and IκB are also reduced. Since Jagged-1is apparently involved in cell differentiation, FGFR-1 signaling isantagonized by effectors which promote differentiation such asγ-interferon, PMA, interleukin-1, and tumor necrosis factor (Friesel etal., 1987, J. Cell Biol. 104:689-696; Hla et al., 1990, Biochem.Biophys. kes. Commun. 167:637-643), and since many of these modifiers ofdifferentiation are involved in NFκβ-mediated signaling (Collins, 1993,Lab. Invest. 68:499-508), it is possible, without wishing to be bound byany particular theory, that Jagged-1-mediated Notch signaling isinvolved in regulating these events. This suggestion is consistent withthe up-regulation of enhancer of split-Groucho, a known component ofNotch signaling (Sun et al., 1996, Development 122:2465-2474). Likewise,connexin, which plays an important role in the formation of tight cellto cell contacts (Pepper and Meda, 1992, J. Cell Physiol. 153:196-205),can be modified during Jagged-1 dependent chord development. Withoutwishing to be bound by theory, it is also interesting that, likeconnexin, the increase in the expression of the type IV collagenasetranscript may be relevant to the differentiation process since it iswell established that proteolytic modification of collagen matrices is acomponent of the migratory phenotype (Lochter et al., 1999, Mol. Biol.Cell 10:271-282) during the process of chord development.

In addition, these alterations in gene expression mediated by Jagged-1may also be involved in directing the formation of a chord-likephenotype during the organization component of the non-terminalendothelial cell differentiation pathway (Xue et al., 1999, Hum. Mol.Genet. 8:723-730). While transmission electron microscopic analysis ofthe chord-like structures revealed prominent interdigitations betweencells with close membrane-membrane apposition, a distinct lumen withinterdigitation of the plasma membrane was not readily observed despitetheir resemblance to the tubular phenotype observed with in vitropopulations of the endothelial cells (see, e.g., Zimrin et al., 1996, J.Biol. Chem. 271:32499-32502). Without wishing to be bound by anyparticular theory, the data disclosed herein suggest that the absence ofreadily visible lumen in the soluble Jagged-1 NIH 3T3 cell transfectantsmay be either a consequence of another gene product, the absence ofappropriate rheologic conditions, or the absence of another genomicrequisite not present in the NIH 3T3 cell. Without wishing to be boundby any particular theory, the data disclosed herein further suggest thatit is likely that another gene product may be responsible for lumenformations since the majority of the CD31-positive chord-like structuresestablished in the soluble Jagged-1 tissue masses in vivo, also do notexhibit evidence of blood flow.

These data are consistent with the recent genetic observation that theJagged-1 null mouse exhibits normal vasculogenesis but an abnormal andearly lethal embryonic angiogenic phenotype including defects in theremodeling of the yolk sac and embryonic vasculature (Xue et al., 1999,Hum. Mol. Genet. 8:723-730). Indeed, the vascular pathology apparent inthe Jagged-1 null mouse (id.) may be related to the inability tomodulate the chord development component of the endothelial celldifferentiation pathway (Zimrin et al., 1996, J. Biol. Chem.271:32499-32502).

Likewise, the observation that mutations in the human Notch-4 gene areresponsible for the formation of CADASIL, a systemic vascular disease(Joutel et al., 1996, Nature 383:707-710) is also consistent with theconcept that Notch signaling is an important component of vascularphysiology in humans. It is also noteworthy that observation relatingthe repression of Jagged-1 function in human endothelial cells to anexaggeration of the ability of FGF but not VEGF to induce sproutformation also correlates well with the role of VEGF but not FGF as amediator of vasculogenesis since the Jagged-1 null mice exhibithemorrhage as a result of the failure to form the large vitelline bloodvessels, a process mediated by angiogenesis (Xue et al., 1999, Hum. Mol.Genet. 8:723-730). Thus, this defect may ultimately involve enhancedendothelial cell sprout formation and a failure of the mutantvasculature to form chords.

The function of the ectodomain of Jagged-1 as a biological responsemodifier is also consistent with the recent observation (Qi et al.,1999, Science 283:91) that the enzymatic function of kuzbanian, an ADAMmetalloprotease gene family member (Rooke et al., 1996, Science273:1227), is required for the activity of the Drosophila Notch ligand,Delta. Although it is not known whether a proteolytic modification ofthe Drosophila Jagged-1 homolog, Serrate (Baker et al., 1990, Science250:1370-1377), requires a similar proteolytic modification, the datadisclosed herein suggest that the ectodomain of Jagged-1 may function inthe absence of its transmembrane domain as an extracellular protein.

The data disclosed herein indicate that Notch-Jagged signaling plays animportant role in neoplasia. For instance, recent data demonstrated thatNotch receptor expression is up-regulated in cervical cancer, Notchmutants can induce neoplastic transformation in the mammary and salivaryglands, and that Notch translocation is associated with human T celllymphoblastic neoplasms (Pear et al., 1996, J. Exp. Med. 183:2283-2291).Further, studies with human cervical carcinoma specimens demonstratethat Jagged-1 is absent in normal cervix, and is overexpressed, alongwith Notch, in malignant cervical adenocarcinoma. The observation thatthe Jagged-1 transcript is present in metaplastic lesions suggests thatit may be involved in early pre-malignant lesion development. Therefore,without wishing to be bound by any particular theory, the data disclosedherein suggest that Jagged-1 may possess a multifaceted role incarcinogenesis by directly influencing cell-fate decisions in theneoplastic cells and by regulating endothelial cell chord developmentduring angiogenesis.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

56 1 1218 PRT Homo sapiens 1 Met Arg Ser Pro Arg Thr Arg Gly Arg Ser GlyArg Pro Leu Ser Leu 1 5 10 15 Leu Leu Ala Leu Leu Cys Ala Leu Arg AlaLys Val Cys Gly Ala Ser 20 25 30 Gly Gln Phe Glu Leu Glu Ile Leu Ser MetGln Asn Val Asn Gly Glu 35 40 45 Leu Gln Asn Gly Asn Cys Cys Gly Gly AlaArg Asn Pro Gly Asp Arg 50 55 60 Lys Cys Thr Arg Asp Glu Cys Asp Thr TyrPhe Lys Val Cys Leu Lys 65 70 75 80 Glu Tyr Gln Ser Arg Val Thr Ala GlyGly Pro Cys Ser Phe Gly Ser 85 90 95 Gly Ser Thr Pro Val Ile Gly Gly AsnThr Phe Asn Leu Lys Ala Ser 100 105 110 Arg Gly Asn Asp Arg Asn Arg IleVal Leu Pro Phe Ser Phe Ala Trp 115 120 125 Pro Arg Ser Tyr Thr Leu LeuVal Glu Ala Trp Asp Ser Ser Asn Asp 130 135 140 Thr Val Gln Pro Asp SerIle Ile Glu Lys Ala Ser His Ser Gly Met 145 150 155 160 Ile Asn Pro SerArg Gln Trp Gln Thr Leu Lys Gln Asn Thr Gly Val 165 170 175 Ala His PheGlu Tyr Gln Ile Arg Val Thr Cys Asp Asp Tyr Tyr Tyr 180 185 190 Gly PheGly Cys Asn Lys Phe Cys Arg Pro Arg Asp Asp Phe Phe Gly 195 200 205 HisTyr Ala Cys Asp Gln Asn Gly Asn Lys Thr Cys Met Glu Gly Trp 210 215 220Met Gly Pro Glu Cys Asn Arg Ala Ile Cys Arg Gln Gly Cys Ser Pro 225 230235 240 Lys His Gly Ser Cys Lys Leu Pro Gly Asp Cys Arg Cys Gln Tyr Gly245 250 255 Trp Gln Gly Leu Tyr Cys Asp Lys Cys Ile Pro His Pro Gly CysVal 260 265 270 His Gly Ile Cys Asn Glu Pro Trp Gln Cys Leu Cys Glu ThrAsn Trp 275 280 285 Gly Gly Gln Leu Cys Asp Lys Asp Leu Asn Tyr Cys GlyThr His Gln 290 295 300 Pro Cys Leu Asn Gly Gly Thr Cys Ser Asn Thr GlyPro Asp Lys Tyr 305 310 315 320 Gln Cys Ser Cys Pro Glu Gly Tyr Ser GlyPro Asn Cys Glu Ile Ala 325 330 335 Glu His Ala Cys Leu Ser Asp Pro CysHis Asn Arg Gly Ser Cys Lys 340 345 350 Glu Thr Ser Leu Gly Phe Glu CysGlu Cys Ser Pro Gly Trp Thr Gly 355 360 365 Pro Thr Cys Ser Thr Asn IleAsp Asp Cys Ser Pro Asn Asn Cys Ser 370 375 380 His Gly Gly Thr Cys GlnAsp Leu Val Asn Gly Phe Lys Cys Val Cys 385 390 395 400 Pro Pro Gln TrpThr Gly Lys Thr Cys Gln Leu Asp Ala Asn Glu Cys 405 410 415 Glu Ala LysPro Cys Val Asn Ala Lys Ser Cys Lys Asn Leu Ile Ala 420 425 430 Ser TyrTyr Cys Asp Cys Leu Pro Gly Trp Met Gly Gln Asn Cys Asp 435 440 445 IleAsn Ile Asn Asp Cys Leu Gly Gln Cys Gln Asn Asp Ala Ser Cys 450 455 460Arg Asp Leu Val Asn Gly Tyr Arg Cys Ile Cys Pro Pro Gly Tyr Ala 465 470475 480 Gly Asp His Cys Glu Arg Asp Ile Asp Glu Cys Ala Ser Asn Pro Cys485 490 495 Leu Asn Gly Gly His Cys Gln Asn Glu Ile Asn Arg Phe Gln CysLeu 500 505 510 Cys Pro Thr Gly Phe Ser Gly Asn Leu Cys Gln Leu Asp IleAsp Tyr 515 520 525 Cys Glu Pro Asn Pro Cys Gln Asn Gly Ala Gln Cys TyrAsn Arg Ala 530 535 540 Ser Asp Tyr Phe Cys Lys Cys Pro Glu Asp Tyr GluGly Lys Asn Cys 545 550 555 560 Ser His Leu Lys Asp His Cys Arg Thr ThrPro Cys Glu Val Ile Asp 565 570 575 Ser Cys Thr Val Ala Met Ala Ser AsnAsp Thr Pro Glu Gly Val Arg 580 585 590 Tyr Ile Ser Ser Asn Val Cys GlyPro His Gly Lys Cys Lys Ser Gln 595 600 605 Ser Gly Gly Lys Phe Thr CysAsp Cys Asn Lys Gly Phe Thr Gly Thr 610 615 620 Tyr Cys His Glu Asn IleAsn Asp Cys Glu Ser Asn Pro Cys Arg Asn 625 630 635 640 Gly Gly Thr CysIle Asp Gly Val Asn Ser Tyr Lys Cys Ile Cys Ser 645 650 655 Asp Gly TrpGlu Gly Ala Tyr Cys Glu Thr Asn Ile Asn Asp Cys Ser 660 665 670 Gln AsnPro Cys His Asn Gly Gly Thr Cys Arg Asp Leu Val Asn Asp 675 680 685 PheTyr Cys Asp Cys Lys Asn Gly Trp Lys Gly Lys Thr Cys His Ser 690 695 700Arg Asp Ser Gln Cys Asp Glu Ala Thr Cys Asn Asn Gly Gly Thr Cys 705 710715 720 Tyr Asp Glu Gly Asp Ala Phe Lys Cys Met Cys Pro Gly Gly Trp Glu725 730 735 Gly Thr Thr Cys Asn Ile Ala Arg Asn Ser Ser Cys Leu Pro AsnPro 740 745 750 Cys His Asn Gly Gly Thr Cys Val Val Asn Gly Glu Ser PheThr Cys 755 760 765 Val Cys Lys Glu Gly Trp Glu Gly Pro Ile Cys Ala GlnAsn Thr Asn 770 775 780 Asp Cys Ser Pro His Pro Cys Tyr Asn Ser Gly ThrCys Val Asp Gly 785 790 795 800 Asp Asn Trp Tyr Arg Cys Glu Cys Ala ProGly Phe Ala Gly Pro Asp 805 810 815 Cys Arg Ile Asn Ile Asn Glu Cys GlnSer Ser Pro Cys Ala Phe Gly 820 825 830 Ala Thr Cys Val Asp Glu Ile AsnGly Tyr Arg Cys Val Cys Pro Pro 835 840 845 Gly His Ser Gly Ala Lys CysGln Glu Val Ser Gly Arg Pro Cys Ile 850 855 860 Thr Met Gly Ser Val IlePro Asp Gly Ala Lys Trp Asp Asp Asp Cys 865 870 875 880 Asn Thr Cys GlnCys Leu Asn Gly Arg Ile Ala Cys Ser Lys Val Trp 885 890 895 Cys Gly ProArg Pro Cys Leu Leu His Lys Gly His Ser Glu Cys Pro 900 905 910 Ser GlyGln Ser Cys Ile Pro Ile Leu Asp Asp Gln Cys Phe Val His 915 920 925 ProCys Thr Gly Val Gly Glu Cys Arg Ser Ser Ser Leu Gln Pro Val 930 935 940Lys Thr Lys Cys Thr Ser Asp Ser Tyr Tyr Gln Asp Asn Cys Ala Asn 945 950955 960 Ile Thr Phe Thr Phe Asn Lys Glu Met Met Ser Pro Gly Leu Thr Thr965 970 975 Glu His Ile Cys Ser Glu Leu Arg Asn Leu Asn Ile Leu Lys AsnVal 980 985 990 Ser Ala Glu Tyr Ser Ile Tyr Ile Ala Cys Glu Pro Ser ProSer Ala 995 1000 1005 Asn Asn Glu Ile His Val Ala Ile Ser Ala Glu AspIle Arg Asp 1010 1015 1020 Asp Gly Asn Pro Ile Lys Glu Ile Thr Asp LysIle Ile Asp Leu 1025 1030 1035 Val Ser Lys Arg Asp Gly Asn Ser Ser LeuIle Ala Ala Val Ala 1040 1045 1050 Glu Val Arg Val Gln Arg Arg Pro LeuLys Asn Arg Thr Asp Phe 1055 1060 1065 Leu Val Pro Leu Leu Ser Ser ValLeu Thr Val Ala Trp Ile Cys 1070 1075 1080 Cys Leu Val Thr Ala Phe TyrTrp Cys Leu Arg Lys Arg Arg Lys 1085 1090 1095 Pro Gly Ser His Thr HisSer Ala Ser Glu Asp Asn Thr Thr Asn 1100 1105 1110 Asn Val Arg Glu GlnLeu Asn Gln Ile Lys Asn Pro Ile Glu Lys 1115 1120 1125 His Gly Ala AsnThr Val Pro Ile Lys Asp Tyr Glu Asn Lys Asn 1130 1135 1140 Ser Lys MetSer Lys Ile Arg Thr His Asn Ser Glu Val Glu Glu 1145 1150 1155 Asp AspMet Asp Lys His Gln Gln Lys Ala Arg Phe Gly Lys Gln 1160 1165 1170 ProAla Tyr Thr Leu Val Asp Arg Glu Glu Lys Pro Pro Asn Gly 1175 1180 1185Thr Pro Thr Lys His Pro Asn Trp Thr Asn Lys Gln Asp Asn Arg 1190 11951200 Asp Leu Glu Ser Ala Gln Ser Leu Asn Arg Met Glu Tyr Ile Val 12051210 1215 2 3657 DNA Homo sapiens 2 atgcgttccc cacggacrcg cggccggtccgggcgccccc taagcctcct gctcgccctg 60 ctctgtgccc tgcgagccaa ggtgtgtggggcctcgggtc agttcgagtt ggagatcctg 120 tccatgcaga acgtgaacgg ggagctgcagaacgggaact gctgcggcgg cgcccggaac 180 ccgggagacc gcaagtgcac ccgcgacgagtgtgacacat acttcaaagt gtgcctcaag 240 gagtatcagt cccgcgtcac ggccggggggccctgcagct tcggctcagg gtccacgcct 300 gtcatcgggg gcaacacctt caacctcaaggccagccgcg gcaacgaccg caaccgcatc 360 gtgctgcctt tcagtttcgc ctggccgaggtcctatacgt tgcttgtgga ggcgtgggat 420 tccagtaatg acaccgttca acctgacagtattattgaaa aggcttctca ctcgggcatg 480 atcaacccca gccggcagtg gcagacgctgaagcagaaca cgggcgttgc ccactttgag 540 tatcagatcc gcgtgacctg tgatgactactactatggct ttggctgyaa taagttctgc 600 cgccccagag atgacttctt tggacactatgcctgtgacc agaatggcaa caaaacttgc 660 atggaaggct ggatgggccc cgaatgtaacagagctattt gccgacaagg ctgcagtcct 720 aagcatgggt cttgcaaact cccaggtgactgcaggtgcc agtayggctg gcaaggcctg 780 tactgtgata agtgcatccc acacccgggatgcgtccacg gcatctgtaa tgagccctgg 840 cagtgcctct gtgagaccaa ctggggcggccagctctgtg acaaagatct caattactgt 900 gggactcatc agccgtgtct caacgggggaacttgtagca acacaggccc tgacaaatat 960 cagtgttcct gccctgaggg gtattcaggacccaactgtg aaattgctga gcacgcctgc 1020 ctctctgatc cctgtcacaa cagaggcagctgtaaggaga cctccctggg ctttgagtgt 1080 gagtgttccc caggctggac cggccccacatgctctacaa acattgatga ctgttctcct 1140 aataactgtt cccacggggg cacctgccaggacctggtta acggatttaa gtgtgtgtgc 1200 cccccacagt ggactgggaa aacgtgccagttagatgcaa atgaatgtga ggccaaacct 1260 tgtgtaaacg ccaaatcctg taagaatctcattgccagct actactgcga ctgtcttccc 1320 ggctggatgg gtcagaattg tgacataaatattaatgact gccttggcca gtgtcagaat 1380 gacgcctcct gtcgggattt ggttaatggttatcgctgta tctgtccacc tggctatgca 1440 ggcgatcact gtgagagaga catcgatgaatgtgccagca acccctgttt gaatgggggt 1500 cactgtcaga atgaaatcaa cagattccagtgtctgtgtc ccactggttt ctctggaaac 1560 ctctgtcagc tggacatcga ttattgtgagcctaatccct gccagaacgg tgcccagtgc 1620 tacaaccgtg ccagtgacta tttctgcaagtgccccgagg actatgaggg caagaactgc 1680 tcacacctga aagaccactg ccgcacgaccccctgtgaag tgattgacag ctgcacagtg 1740 gccatggctt ccaacgacac acctgaaggggtgcggtata tttcctccaa cgtctgtggt 1800 cctcacggga agtgcaagag tcagtcgggaggcaaattca cctgtgactg taacaaaggc 1860 ttcacgggaa catactgcca tgaaaatattaatgactgtg agagcaaccc ttgtagaaac 1920 ggtggcactt gcatcgatgg tgtcaactcctacaagtgca tctgtagtga cggctgggag 1980 ggggcctact gtgaaaccaa tattaatgactgcagccaga acccctgcca caatgggggc 2040 acgtgtcgcg acctggtcaa tgacttctactgtgactgta aaaatgggtg gaaaggaaag 2100 acctgccact cacgtgacag tcagtgtgatgaggccacgt gcaacaacgg tggcacctgc 2160 tatgatgagg gggatgcttt taagtgcatgtgtcctggcg gctgggaagg aacaacctgt 2220 aacatagccc gaaacagtag ctgcctgcccaacccctgcc ataatggggg cacatgtgtg 2280 gtcaacggcg agtcctttac gtgcgtctgcaaggaaggct gggaggggcc catctgtgct 2340 cagaatacca atgactgcag ccctcatccctgttacaaca gcggcacctg tgtggatgga 2400 gacaactggt accggtgcga atgtgccccgggttttgctg ggcccgactg cagaataaac 2460 atcaatgaat gccagtcttc accttgtgcctttggagcga cctgtgtgga tgagatcaat 2520 ggctaccggt gtgtctgccc tccagggcacagtggtgcca agtgccagga agtttcaggg 2580 agaccttgca tcaccatggg gagtgtgataccagatgggg ccaaatggga tgatgactgt 2640 aatacctgcc agtgcctgaa tggacggatcgcctgctcaa aggtctggtg tggccctcga 2700 ccttgcctgc tccacaaagg gcacagcgagtgccccagcg ggcagagctg catccccatc 2760 ctggacgacc agtgcttcgt ccacccctgcactggtgtgg gcgagtgtcg gtcttccagt 2820 ctccagccgg tgaagacaaa gtgcacctctgactcctatt accaggataa ctgtgcgaac 2880 atcacattta cctttaacaa ggagatgatgtcaccaggtc ttactacgga gcacatttgc 2940 agtgaattga ggaatttgaa tattttgaagaatgtttccg ctgaatattc aatctacatc 3000 gcttgcgagc cttccccttc agcgaacaatgaaatacatg tggccatttc tgctgaagat 3060 atacgggatg atgggaaccc gatcaaggaaatcactgaca aaataatcga tcttgttagt 3120 aaacgtgatg gaaacagctc gctgattgctgccgttgcag aagtaagagt tcagaggcgg 3180 cctctgaaga acagaacaga tttccttgttcccttgctga gctctgtctt aactgtggct 3240 tggatctgtt gcttggtgac ggccttctactggtgcctgc ggaagcggcg gaagccgggc 3300 agccacacac actcagcctc tgaggacaacaccaccaaca acgtgcggga gcagctgaac 3360 cagatcaaaa accccattga gaaacatggggccaacacgg tccccatcaa ggattacgag 3420 aacaagaact ccaaaatgtc taaaataaggacacacaatt ctgaagtaga agaggacgac 3480 atggacaaac accagcagaa agcccggtttggcaagcagc cggcgtatac gctggtagac 3540 agagaagaga agccccccaa cggcacgccgacaaaacacc caaactggac aaacaaacag 3600 gacaacagag acttggaaag tgcccagagcttaaaccgaa tggagtacat cgtatag 3657 3 22 DNA Artificial Sequence PCRprimer 3 gcgcaagctt tttttttttt cg 22 4 18 DNA Artificial Sequence PCRprimer 4 gagaccgtga agatactt 18 5 20 DNA Artificial Sequence PCR primer5 ccgactgcag aataaacatc 20 6 20 DNA Artificial Sequence PCR primer 6ttggatctgg ttcagctgct 20 7 20 DNA Artificial Sequence PCR primer 7ttcagtgacg gccactgtga 20 8 20 DNA Artificial Sequence PCR primer 8cacgtacatg aagtgcagct 20 9 20 DNA Artificial Sequence PCR primer 9tgagtaggct ccatccagtc 20 10 20 DNA Artificial Sequence PCR primer 10tggtgtcagg tagggatgct 20 11 24 DNA Artificial Sequence PCR primer 11ccacccatgg caaattccat ggca 24 12 24 DNA Artificial Sequence PCR primer12 tctagacggc aggtcaggtc cacc 24 13 36 DNA Artificial Sequence PCRprimer 13 gactatgcga attcggatcc gtcgacgcca ccatgg 36 14 20 DNAArtificial Sequence PCR primer 14 caagttcccc cgttgagaca 20 15 65 DNAArtificial Sequence PCR primer 15 gcatagtcct cgagttacaa gtcttcttcagaaataagct tttgttctac gatgtactcc 60 attcg 65 16 20 DNA ArtificialSequence PCR primer 16 atggacaaac accagcagaa 20 17 3201 DNA Homo sapiens17 atgcgttccc cacggacrcg cggccggtcc gggcgccccc taagcctcct gctcgccctg 60ctctgtgccc tgcgagccaa ggtgtgtggg gcctcgggtc agttcgagtt ggagatcctg 120tccatgcaga acgtgaacgg ggagctgcag aacgggaact gctgcggcgg cgcccggaac 180ccgggagacc gcaagtgcac ccgcgacgag tgtgacacat acttcaaagt gtgcctcaag 240gagtatcagt cccgcgtcac ggccgggggg ccctgcagct tcggctcagg gtccacgcct 300gtcatcgggg gcaacacctt caacctcaag gccagccgcg gcaacgaccg caaccgcatc 360gtgctgcctt tcagtttcgc ctggccgagg tcctatacgt tgcttgtgga ggcgtgggat 420tccagtaatg acaccgttca acctgacagt attattgaaa aggcttctca ctcgggcatg 480atcaacccca gccggcagtg gcagacgctg aagcagaaca cgggcgttgc ccactttgag 540tatcagatcc gcgtgacctg tgatgactac tactatggct ttggctgyaa taagttctgc 600cgccccagag atgacttctt tggacactat gcctgtgacc agaatggcaa caaaacttgc 660atggaaggct ggatgggccc cgaatgtaac agagctattt gccgacaagg ctgcagtcct 720aagcatgggt cttgcaaact cccaggtgac tgcaggtgcc agtayggctg gcaaggcctg 780tactgtgata agtgcatccc acacccggga tgcgtccacg gcatctgtaa tgagccctgg 840cagtgcctct gtgagaccaa ctggggcggc cagctctgtg acaaagatct caattactgt 900gggactcatc agccgtgtct caacggggga acttgtagca acacaggccc tgacaaatat 960cagtgttcct gccctgaggg gtattcagga cccaactgtg aaattgctga gcacgcctgc 1020ctctctgatc cctgtcacaa cagaggcagc tgtaaggaga cctccctggg ctttgagtgt 1080gagtgttccc caggctggac cggccccaca tgctctacaa acattgatga ctgttctcct 1140aataactgtt cccacggggg cacctgccag gacctggtta acggatttaa gtgtgtgtgc 1200cccccacagt ggactgggaa aacgtgccag ttagatgcaa atgaatgtga ggccaaacct 1260tgtgtaaacg ccaaatcctg taagaatctc attgccagct actactgcga ctgtcttccc 1320ggctggatgg gtcagaattg tgacataaat attaatgact gccttggcca gtgtcagaat 1380gacgcctcct gtcgggattt ggttaatggt tatcgctgta tctgtccacc tggctatgca 1440ggcgatcact gtgagagaga catcgatgaa tgtgccagca acccctgttt gaatgggggt 1500cactgtcaga atgaaatcaa cagattccag tgtctgtgtc ccactggttt ctctggaaac 1560ctctgtcagc tggacatcga ttattgtgag cctaatccct gccagaacgg tgcccagtgc 1620tacaaccgtg ccagtgacta tttctgcaag tgccccgagg actatgaggg caagaactgc 1680tcacacctga aagaccactg ccgcacgacc ccctgtgaag tgattgacag ctgcacagtg 1740gccatggctt ccaacgacac acctgaaggg gtgcggtata tttcctccaa cgtctgtggt 1800cctcacggga agtgcaagag tcagtcggga ggcaaattca cctgtgactg taacaaaggc 1860ttcacgggaa catactgcca tgaaaatatt aatgactgtg agagcaaccc ttgtagaaac 1920ggtggcactt gcatcgatgg tgtcaactcc tacaagtgca tctgtagtga cggctgggag 1980ggggcctact gtgaaaccaa tattaatgac tgcagccaga acccctgcca caatgggggc 2040acgtgtcgcg acctggtcaa tgacttctac tgtgactgta aaaatgggtg gaaaggaaag 2100acctgccact cacgtgacag tcagtgtgat gaggccacgt gcaacaacgg tggcacctgc 2160tatgatgagg gggatgcttt taagtgcatg tgtcctggcg gctgggaagg aacaacctgt 2220aacatagccc gaaacagtag ctgcctgccc aacccctgcc ataatggggg cacatgtgtg 2280gtcaacggcg agtcctttac gtgcgtctgc aaggaaggct gggaggggcc catctgtgct 2340cagaatacca atgactgcag ccctcatccc tgttacaaca gcggcacctg tgtggatgga 2400gacaactggt accggtgcga atgtgccccg ggttttgctg ggcccgactg cagaataaac 2460atcaatgaat gccagtcttc accttgtgcc tttggagcga cctgtgtgga tgagatcaat 2520ggctaccggt gtgtctgccc tccagggcac agtggtgcca agtgccagga agtttcaggg 2580agaccttgca tcaccatggg gagtgtgata ccagatgggg ccaaatggga tgatgactgt 2640aatacctgcc agtgcctgaa tggacggatc gcctgctcaa aggtctggtg tggccctcga 2700ccttgcctgc tccacaaagg gcacagcgag tgccccagcg ggcagagctg catccccatc 2760ctggacgacc agtgcttcgt ccacccctgc actggtgtgg gcgagtgtcg gtcttccagt 2820ctccagccgg tgaagacaaa gtgcacctct gactcctatt accaggataa ctgtgcgaac 2880atcacattta cctttaacaa ggagatgatg tcaccaggtc ttactacgga gcacatttgc 2940agtgaattga ggaatttgaa tattttgaag aatgtttccg ctgaatattc aatctacatc 3000gcttgcgagc cttccccttc agcgaacaat gaaatacatg tggccatttc tgctgaagat 3060atacgggatg atgggaaccc gatcaaggaa atcactgaca aaataatcga tcttgttagt 3120aaacgtgatg gaaacagctc gctgattgct gccgttgcag aagtaagagt tcagaggcgg 3180cctctgaaga acagaacaga t 3201 18 1067 PRT Homo sapiens 18 Met Arg Ser ProArg Thr Arg Gly Arg Ser Gly Arg Pro Leu Ser Leu 1 5 10 15 Leu Leu AlaLeu Leu Cys Ala Leu Arg Ala Lys Val Cys Gly Ala Ser 20 25 30 Gly Gln PheGlu Leu Glu Ile Leu Ser Met Gln Asn Val Asn Gly Glu 35 40 45 Leu Gln AsnGly Asn Cys Cys Gly Gly Ala Arg Asn Pro Gly Asp Arg 50 55 60 Lys Cys ThrArg Asp Glu Cys Asp Thr Tyr Phe Lys Val Cys Leu Lys 65 70 75 80 Glu TyrGln Ser Arg Val Thr Ala Gly Gly Pro Cys Ser Phe Gly Ser 85 90 95 Gly SerThr Pro Val Ile Gly Gly Asn Thr Phe Asn Leu Lys Ala Ser 100 105 110 ArgGly Asn Asp Arg Asn Arg Ile Val Leu Pro Phe Ser Phe Ala Trp 115 120 125Pro Arg Ser Tyr Thr Leu Leu Val Glu Ala Trp Asp Ser Ser Asn Asp 130 135140 Thr Val Gln Pro Asp Ser Ile Ile Glu Lys Ala Ser His Ser Gly Met 145150 155 160 Ile Asn Pro Ser Arg Gln Trp Gln Thr Leu Lys Gln Asn Thr GlyVal 165 170 175 Ala His Phe Glu Tyr Gln Ile Arg Val Thr Cys Asp Asp TyrTyr Tyr 180 185 190 Gly Phe Gly Cys Asn Lys Phe Cys Arg Pro Arg Asp AspPhe Phe Gly 195 200 205 His Tyr Ala Cys Asp Gln Asn Gly Asn Lys Thr CysMet Glu Gly Trp 210 215 220 Met Gly Pro Glu Cys Asn Arg Ala Ile Cys ArgGln Gly Cys Ser Pro 225 230 235 240 Lys His Gly Ser Cys Lys Leu Pro GlyAsp Cys Arg Cys Gln Tyr Gly 245 250 255 Trp Gln Gly Leu Tyr Cys Asp LysCys Ile Pro His Pro Gly Cys Val 260 265 270 His Gly Ile Cys Asn Glu ProTrp Gln Cys Leu Cys Glu Thr Asn Trp 275 280 285 Gly Gly Gln Leu Cys AspLys Asp Leu Asn Tyr Cys Gly Thr His Gln 290 295 300 Pro Cys Leu Asn GlyGly Thr Cys Ser Asn Thr Gly Pro Asp Lys Tyr 305 310 315 320 Gln Cys SerCys Pro Glu Gly Tyr Ser Gly Pro Asn Cys Glu Ile Ala 325 330 335 Glu HisAla Cys Leu Ser Asp Pro Cys His Asn Arg Gly Ser Cys Lys 340 345 350 GluThr Ser Leu Gly Phe Glu Cys Glu Cys Ser Pro Gly Trp Thr Gly 355 360 365Pro Thr Cys Ser Thr Asn Ile Asp Asp Cys Ser Pro Asn Asn Cys Ser 370 375380 His Gly Gly Thr Cys Gln Asp Leu Val Asn Gly Phe Lys Cys Val Cys 385390 395 400 Pro Pro Gln Trp Thr Gly Lys Thr Cys Gln Leu Asp Ala Asn GluCys 405 410 415 Glu Ala Lys Pro Cys Val Asn Ala Lys Ser Cys Lys Asn LeuIle Ala 420 425 430 Ser Tyr Tyr Cys Asp Cys Leu Pro Gly Trp Met Gly GlnAsn Cys Asp 435 440 445 Ile Asn Ile Asn Asp Cys Leu Gly Gln Cys Gln AsnAsp Ala Ser Cys 450 455 460 Arg Asp Leu Val Asn Gly Tyr Arg Cys Ile CysPro Pro Gly Tyr Ala 465 470 475 480 Gly Asp His Cys Glu Arg Asp Ile AspGlu Cys Ala Ser Asn Pro Cys 485 490 495 Leu Asn Gly Gly His Cys Gln AsnGlu Ile Asn Arg Phe Gln Cys Leu 500 505 510 Cys Pro Thr Gly Phe Ser GlyAsn Leu Cys Gln Leu Asp Ile Asp Tyr 515 520 525 Cys Glu Pro Asn Pro CysGln Asn Gly Ala Gln Cys Tyr Asn Arg Ala 530 535 540 Ser Asp Tyr Phe CysLys Cys Pro Glu Asp Tyr Glu Gly Lys Asn Cys 545 550 555 560 Ser His LeuLys Asp His Cys Arg Thr Thr Pro Cys Glu Val Ile Asp 565 570 575 Ser CysThr Val Ala Met Ala Ser Asn Asp Thr Pro Glu Gly Val Arg 580 585 590 TyrIle Ser Ser Asn Val Cys Gly Pro His Gly Lys Cys Lys Ser Gln 595 600 605Ser Gly Gly Lys Phe Thr Cys Asp Cys Asn Lys Gly Phe Thr Gly Thr 610 615620 Tyr Cys His Glu Asn Ile Asn Asp Cys Glu Ser Asn Pro Cys Arg Asn 625630 635 640 Gly Gly Thr Cys Ile Asp Gly Val Asn Ser Tyr Lys Cys Ile CysSer 645 650 655 Asp Gly Trp Glu Gly Ala Tyr Cys Glu Thr Asn Ile Asn AspCys Ser 660 665 670 Gln Asn Pro Cys His Asn Gly Gly Thr Cys Arg Asp LeuVal Asn Asp 675 680 685 Phe Tyr Cys Asp Cys Lys Asn Gly Trp Lys Gly LysThr Cys His Ser 690 695 700 Arg Asp Ser Gln Cys Asp Glu Ala Thr Cys AsnAsn Gly Gly Thr Cys 705 710 715 720 Tyr Asp Glu Gly Asp Ala Phe Lys CysMet Cys Pro Gly Gly Trp Glu 725 730 735 Gly Thr Thr Cys Asn Ile Ala ArgAsn Ser Ser Cys Leu Pro Asn Pro 740 745 750 Cys His Asn Gly Gly Thr CysVal Val Asn Gly Glu Ser Phe Thr Cys 755 760 765 Val Cys Lys Glu Gly TrpGlu Gly Pro Ile Cys Ala Gln Asn Thr Asn 770 775 780 Asp Cys Ser Pro HisPro Cys Tyr Asn Ser Gly Thr Cys Val Asp Gly 785 790 795 800 Asp Asn TrpTyr Arg Cys Glu Cys Ala Pro Gly Phe Ala Gly Pro Asp 805 810 815 Cys ArgIle Asn Ile Asn Glu Cys Gln Ser Ser Pro Cys Ala Phe Gly 820 825 830 AlaThr Cys Val Asp Glu Ile Asn Gly Tyr Arg Cys Val Cys Pro Pro 835 840 845Gly His Ser Gly Ala Lys Cys Gln Glu Val Ser Gly Arg Pro Cys Ile 850 855860 Thr Met Gly Ser Val Ile Pro Asp Gly Ala Lys Trp Asp Asp Asp Cys 865870 875 880 Asn Thr Cys Gln Cys Leu Asn Gly Arg Ile Ala Cys Ser Lys ValTrp 885 890 895 Cys Gly Pro Arg Pro Cys Leu Leu His Lys Gly His Ser GluCys Pro 900 905 910 Ser Gly Gln Ser Cys Ile Pro Ile Leu Asp Asp Gln CysPhe Val His 915 920 925 Pro Cys Thr Gly Val Gly Glu Cys Arg Ser Ser SerLeu Gln Pro Val 930 935 940 Lys Thr Lys Cys Thr Ser Asp Ser Tyr Tyr GlnAsp Asn Cys Ala Asn 945 950 955 960 Ile Thr Phe Thr Phe Asn Lys Glu MetMet Ser Pro Gly Leu Thr Thr 965 970 975 Glu His Ile Cys Ser Glu Leu ArgAsn Leu Asn Ile Leu Lys Asn Val 980 985 990 Ser Ala Glu Tyr Ser Ile TyrIle Ala Cys Glu Pro Ser Pro Ser Ala 995 1000 1005 Asn Asn Glu Ile HisVal Ala Ile Ser Ala Glu Asp Ile Arg Asp 1010 1015 1020 Asp Gly Asn ProIle Lys Glu Ile Thr Asp Lys Ile Ile Asp Leu 1025 1030 1035 Val Ser LysArg Asp Gly Asn Ser Ser Leu Ile Ala Ala Val Ala 1040 1045 1050 Glu ValArg Val Gln Arg Arg Pro Leu Lys Asn Arg Thr Asp 1055 1060 1065 19 54 DNAArtificial Sequence PCR primer 19 gactatgcga attcggatcc gtcgacgccaccatgggttc cccacggaca cgcg 54 20 20 DNA Artificial Sequence PCR primer20 caagttcccc cgttgagaca 20 21 20 DNA Artificial Sequence PCR primer 21atggacaaac accagcagaa 20 22 65 DNA Artificial Sequence PCR primer 22tagtgctcga gctattacaa gtcttcttca gaaataagct tttgttcatc tgttctgttc 60ttcag 65 23 44 DNA Artificial Sequence PCR primer 23 tttggatttgctggtgcagt acaactaggc ttaataggga catg 44 24 37 DNA Artificial SequencePCR primer 24 tccctattaa gcctagttgt actgcaccag caaatcc 37 25 42 DNAArtificial Sequence PCR primer 25 tttctgctcg aattcaagct tctaacgatgtacggggaca tg 42 26 35 DNA Artificial Sequence PCR primer 26 tccccgtacatcgttagaag cttgaattcg agcag 35 27 23 DNA Artificial Sequence PCR primer27 ggatttgctg gtgcagtaca act 23 28 23 DNA Artificial Sequence PCR primer28 ctgctcgaat tcaagcttct aac 23 29 18 DNA Artificial Sequence Jaggedantisense oligomer 29 tggggaccgc atcgctgc 18 30 18 DNA ArtificialSequence Jagged sense oligomer 30 gcagcgatgc ggtcccca 18 31 18 DNAArtificial Sequence 3′ Jagged antisense oligomer 31 gaatcaaggc tcccctag18 32 18 DNA Artificial Sequence Mutated 5′ Jagged antisense oligomer 32tgcggtcccc aacggtgg 18 33 4 PRT Homo sapiens 33 Pro Glu Ser Thr 1 34 10DNA Mus musculus 34 tggatcagtc 10 35 10 DNA Mus musculus 35 taaagaggcc10 36 10 DNA Mus musculus 36 cctgatcttt 10 37 10 DNA Mus musculus 37tgtaacagga 10 38 10 DNA Mus musculus 38 tctgtgcacc 10 39 10 DNA Musmusculus 39 ccaaataaaa 10 40 10 DNA Mus musculus 40 ctaataaaag 10 41 10DNA Mus musculus 41 gccaagggtc 10 42 10 DNA Mus musculus 42 gtctgctgat10 43 10 DNA Mus musculus 43 aaggaagaga 10 44 10 DNA Mus musculus 44tgaaataaac 10 45 10 DNA Mus musculus 45 caccaccaca 10 46 10 DNA Musmusculus 46 cctcagcctg 10 47 10 DNA Mus musculus 47 ctctgactta 10 48 10DNA Mus musculus 48 gtgggcgtgt 10 49 10 DNA Mus musculus 49 tccttggggg10 50 10 DNA Mus musculus 50 cgcctgctag 10 51 10 DNA Mus musculus 51aaaaaaaaaa 10 52 10 DNA Mus musculus 52 aagcagaagg 10 53 10 DNA Musmusculus 53 caggactccg 10 54 10 DNA Mus musculus 54 gaagcaggac 10 55 10DNA Mus musculus 55 ggatatgtgg 10 56 10 DNA Mus musculus 56 gttctgattg10

What is claimed is:
 1. An isolated nucleic acid encoding a solubleJagged protein consisting of the sequence from nucleotide number 1 tonucleotide number 3201 of SEQ ID NO:2.
 2. A vector comprising theisolated nucleic acid of claim
 1. 3. An isolated nucleic acid encoding asoluble Jagged protein, said soluble Jagged protein consisting of theamino acid sequence SEQ ID NO:18.
 4. A vector comprising the isolatednucleic acid of claim
 3. 5. An isolated chimeric nucleic acid encoding atag polypeptide covalently linked to a soluble jagged protein, whereinsaid soluble jagged protein is encoded by a nucleic acid sequenceconsisting of nucleotide number 1 to nucleotide number 3201 of SEQ IDNO:2.
 6. The isolated nucleic acid of claim 5, wherein said tagpolypeptide is selected from the group consisting of a myc tagpolypeptide, a myc-pyruvate kinase tag polypeptide, aglutathione-S-transferase tag polypeptide, a maltose binding tagpolypeptide, green fluorescence protein tag polypeptide, an alkalinephosphatase tag polypeptide, a His6 tag polypeptide, an influenza virushemagglutinin tag polypeptide, and a maltose binding protein tagpolypeptide.
 7. The isolated nucleic acid of claim 6, wherein said tagpolypeptide is a myc tag polypeptide.
 8. An isolated nucleic acidcomprising a promoter/regulatory sequence operably linked to a nucleicacid encoding a soluble jagged protein, wherein said soluble jaggedprotein is encoded by a nucleic acid sequence consisting of the sequencefrom nucleotide number 1 to nucleotide number 3201 of SEQ ID NO:2.
 9. Arecombinant cell comprising the isolated nucleic acid of claim
 1. 10. Arecombinant cell comprising the isolated nucleic acid of claim
 3. 11. Apharmaceutical composition comprising a therapeutically effective amountof an isolated nucleic acid encoding a soluble Jagged polypeptide,wherein said isolated nucleic acid consists of the sequence of SEQ IDNO:17, in a pharmaceutically acceptable carrier.